abstract
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although microrna-144-3p (mirna-144-3p) has been shown to suppress tumor proliferation and invasion, its function in intracerebral hemorrhage (ich)-induced secondary brain injury (sbi) remains unclear. thus, this study was designed to investigate the role of mirna-144-3p in ich. to accomplish this, we used adult male sprague-dawley rats to establish an in vivo ich model by injecting autologous blood, while cultured primary rat cortical neurons were exposed to oxyhemoglobin (oxyhb) to mimic ich in vitro . to examine the role of mirna-144-3p in ich-induced sbi, we used an mirna-144-3p mimic and inhibitor both in vivo and in vitro . following ich induction, we found mirna-144-3p expression to increase. additionally, we predicted the formyl peptide receptor 2 (fpr2) to be a potential mirna-144-3p target, which we validated experimentally, with fpr2 expression downregulated when mirna-144-3p was upregulated. furthermore, elevated mirna-144-3p levels aggravated brain edema and neurobehavioral disorders and induced neuronal apoptosis via the downregulation of fpr2 both in vivo and in vitro . we suspected that these beneficial effects provided by fpr2 were associated with the pi3k/akt pathway. we validated this finding by overexpressing fpr2 while inhibiting pi3k/akt in vitro and in vivo . in conclusion, mirna-144-3p aggravated ich-induced sbi by targeting and downregulating fpr2, thereby contributing to neurological dysfunction and neural apoptosis via pi3k/akt pathway activation. these findings suggest that inhibiting mirna-144-3p may offer an effective approach to attenuating brain damage incurred after ich and a potential therapy to improve ich-induced sbi.
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a mutation located in the 5'-untranslated region (5'-utr) of the nerve-specific connexin-32 mrna, previously found in a family with charcot-marie-tooth disease (cmtx), was analyzed for its effect on the expression of a reporter gene (luciferase) in transgenic mice and in transfected cells. whereas both mutant and wild-type genes appeared to be transcribed and spliced efficiently, no luciferase was detected from the mutant in either system, suggesting that the mutation affects translation of the mrna. when the 5'-utr of nerve-specific connexin-32 mrna was inserted between the two genes of a bicistronic vector and transfected into various cell lines, expression of the second gene was significantly increased. because the mutant did not facilitate translation of the second gene in the bicistronic mrna system, this result suggested that the cmtx mutation abolished function of an internal ribosome entry site (ires) in the 5'-utr of the wild-type connexin-32 mrna. the cmtx phenotype of the mutant 5'-utr further suggested that the wild-type ires was essential for the translation of the connexin-32 mrna in nerve cells. in addition, other sequence elements of the connexin-32 ires were characterized by mutation analysis. a mutation in either of the first two elements investigated showed loss of ires function, whereas mutation of a third element showed gain of function.
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background micrornas (mirnas) are short, non-coding rnas (~22 nt) that play important roles in the pathogenesis of human diseases by negatively regulating gene expression. although mir-196a has been implicated in several other cancers, its role in non-small cell lung cancer (nsclc) is unknown. the aim of the present study was to examine the expression pattern of mir-196a in nsclc and its clinical significance, as well as its biological role in tumor progression. methods expression of mir-196a was analyzed in 34 nsclc tissues and five nsclc cell lines by quantitative reverse-transcription polymerase chain reaction (qrt-pcr). the effect of dna methylation on mir-196a expression was investigated by 5-aza-2-deoxy-cytidine treatment and bisulfite sequencing. the effect of mir-196a on proliferation was evaluated by mtt and colony formation assays, and cell migration and invasion were evaluated by transwell assays. analysis of target protein expression was determined by western blotting. luciferase reporter plasmids were constructed to confirm the action of mir-196a on downstream target genes, including hoxa5. differences between the results were tested for significance using student's t-test (two-tailed). results mir-196a was highly expressed both in nsclc samples and cell lines compared with their corresponding normal counterparts, and the expression of mir-196a may be affected by dna demethylation. higher expression of mir-196a in nsclc tissues was associated with a higher clinical stage, and also correlated with nsclc lymph-node metastasis. in vitro functional assays demonstrated that modulation of mir-196a expression affected nsclc cell proliferation, migration and invasion. our analysis showed that mir-196a suppressed the expression of hoxa5 both at the mrna and protein levels, and luciferase assays confirmed that mir-196a directly bound to the 3'untranslated region of hoxa5. knockdown of hoxa5 expression in a549 cells using rnai was shown to promote nsclc cell proliferation, migration and invasion. finally, we observed an inverse correlation between hoxa5 and mir-196a expression in nsclc tissues. conclusions our findings indicate that mir-196a is significantly up-regulated in nsclc tissues, and regulates nsclc cell proliferation, migration and invasion, partially via the down-regulation of hoxa5. thus, mir-196a may represent a potential therapeutic target for nsclc intervention.
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here we describe a set of endogenous short interfering rnas (sirnas) in arabidopsis, some of which direct the cleavage of endogenous mrnas. these sirnas correspond to both sense and antisense strands of a noncoding rna (at2g27400) that apparently is converted to double-stranded rna and then processed in 21 nt increments. these sirnas differ from previously described regulatory small rnas in two respects. first, they require components of the cosuppression pathway (rdr6 and sgs3) and also components of the microrna (mirna) pathway (ago1, dcl1, hen1, and hyl1) but not components needed for heterochromatic sirnas (dcl3 and rdr2), another class of endogenous plant sirnas. second, these sirnas repress the expression of genes that have little overall resemblance to the genes from which they originate, a characteristic previously reported only for mirnas. the identification of this silencing pathway provides yet another dimension to posttranscriptional mrna regulation in plants.
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in saccharomyces cerevisiae, seven snrnas (snr3, 4, 5, 8, 9, 10 and 17) are retained in the nucleus under conditions in which nucleoplasmic rnas are lost, and may be nucleolar. all of these snrnas show properties consistent with hydrogen bonding to pre-ribosomal rnas; snr5 and 8 with 20s pre-rrna, snr3, 4, 10 and 17 with 35s pre-rrna and snr9 with 20-35s rna. strains lacking snr10 are impaired in growth and specifically defective in the processing of 35s rna. processing is slowed, leading to 35s rna accumulation and most cleavage occurs, not at the normal sites, but at sites which in wild-type strains are used for subsequent steps in rrna maturation.
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micrornas are short, endogenous non-coding rnas that act as post-transcriptional modulators of gene expression. important functions for micrornas have been found in the regulation of development, cellular proliferation and differentiation, while perturbed mirna expression patterns have been observed in many human cancers. here we present a method for specific inhibition of mirna function through interaction with lna-modified antisense oligonucleotides and report the specificity of this application. we show that lna-modified oligonucleotides can inhibit exogenously introduced mirnas with high specificity using a heterologous reporter assay, and furthermore demonstrate their ability to inhibit an endogenous mirna in drosophila melanogaster cells, leading to up-regulation of the cognate target protein. the method shows stoichiometric and reliable inhibition of the targeted mirna and can thus be applied to studies of mirna functions and validation of putative target genes.
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fsh plays an essential role in processes involved in human reproduction, including spermatogenesis and the ovarian cycle. while the transcriptional regulatory mechanisms underlying its synthesis and secretion have been extensively studied, little is known about its posttranscriptional regulation. a bioinformatics analysis from our group indicated that a microrna (mirna; mir-361-3p) could regulate fsh secretion by potentially targeting the fshb subunit. herein, we sought to confirm these findings by investigating the mir-361-3p-mediated regulation of fsh production in primary pig anterior pituitary cells. gonadotropin-releasing hormone (gnrh) treatment resulted in an increase in fshb synthesis at both the mrna, protein/hormone level, along with a significant decrease in mir-361-3p and its precursor (pre-mir-361) levels in time- and dose-dependent manner. using the dual-luciferase assay, we confirmed that mir-361-3p directly targets fshb. additionally, overexpression of mir-361-3p using mimics significantly decreased the fshb production at both the mrna and protein levels, with a reduction in both protein synthesis and secretion. conversely, both synthesis and secretion were significantly increased following mir-361-3p blockade. to confirm that mir-361-3p targets fshb, we designed fsh-targeted sirnas, and co-transfected anterior pituitary cells with both the sirna and mir-361-3p inhibitors. our results indicated that the sirna blocked the mir-361-3p inhibitor-mediated upregulation of fsh, while no significant effect on non-target expression. taken together, our results demonstrate that mir-361-3p negatively regulates fsh synthesis and secretion by targeting fshb, which provides more functional evidence that a mirna is involved in the direct regulation of fsh.
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progressive hearing loss is common in the human population, but little is known about the molecular basis. we report a new n-ethyl-n-nitrosurea (enu)-induced mouse mutant, diminuendo, with a single base change in the seed region of mirn96. heterozygotes show progressive loss of hearing and hair cell anomalies, whereas homozygotes have no cochlear responses. most micrornas are believed to downregulate target genes by binding to specific sites on their mrnas, so mutation of the seed should lead to target gene upregulation. microarray analysis revealed 96 transcripts with significantly altered expression in homozygotes; notably, slc26a5, ocm, gfi1, ptprq and pitpnm1 were downregulated. hypergeometric p-value analysis showed that hundreds of genes were upregulated in mutants. different genes, with target sites complementary to the mutant seed, were downregulated. this is the first microrna found associated with deafness, and diminuendo represents a model for understanding and potentially moderating progressive hair cell degeneration in hearing loss more generally.
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nodal is a member of the transforming growth factor-β superfamily that plays crucial roles during embryogenesis. recently, we have reported that nodal inhibits trophoblast cell proliferation, migration and invasion, but induces apoptosis in the human placenta. in this study, we examined the regulation of nodal by micrornas. in silico analysis of nodal 3'utr revealed a potential binding site for mir-378a-5p. in luciferase reporter assays, we found that mir-378a-5p suppressed the luciferase activity of a reporter plasmid containing nodal 3'utr but this suppressive effect was completely abolished when the predicted target site was mutated. western blot analysis showed that mir-378a-5p decreased whereas anti-mir-378a-5p increased nodal protein levels. these results indicate that mir-378a-5p targets nodal 3'utr to repress its expression. stable transfection of the mir-378a-5p precursor, mir-378a, into htr8/svneo cells enhanced cell survival, proliferation, migration and invasion. transient transfection of mature mir-378a-5p mimic, and to a lesser extent, sirna targeting nodal, produced similar effects. however, anti-mir-378a-5p inhibited cell migration and invasion. in addition, overexpression of nodal reversed the invasion-promoting effect of mir-378a-5p. furthermore, mir-378a-5p enhanced, whereas anti-mir-378a-5p suppressed, the outgrowth and spreading of extravillous trophoblast cells in first trimester placental explants. finally, mir-378a-5p was detected in human placenta throughout different stages of gestation and in preterm pregnancies, placental mir-378a-5p levels were lower in preeclamptic patients than in healthy controls. taken together, these findings strongly suggest that mir-378a-5p plays an important role in human placental development by regulating trophoblast cell growth, survival, migration and invasion, and that mir-378a-5p exerts these effects, at least in part, through the suppression of nodal expression.
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background micrornas (mirnas) are endogenous, small non-coding rnas that play important roles in multiple biological processes. mir-20b has been reported to participate in breast cancer tumorigenic progression, however, the functional roles are still unclear and under debating. the aim of this study is to explicit the molecular mechanism of mir-20b underlying breast cancer tumorigenesis. results in the present study, we showed that mir-20b was overexpressed in human breast cancer tissues and cell lines compared with paired adjacent normal tissues and normal cell lines, respectively. we identified pten, a well-known tumor suppressor, as the functional downstream target of mir-20b. luciferase assays confirmed that mir-20b could directly bind to the 3' untranslated region(utr) of pten and suppress translation. alteration of mir-20b expression changed pten protein level but not mrna expression in zr-75-30 and mcf-7 breast cancer cells, suggesting mir-20b regulates pten gene expression at the posttranscriptional level. furthermore, upregulation of mir-20b significantly promoted the proliferation, colony formation and dna synthesis of zr-75-30 and mcf-7 breast cancer cells. conversely, knockdown of mir-20b expression inhibited the growth of breast cancer cells in vitro and in vivo. conclusion dysregulation of mir-20b plays critical roles in the breast cancer tumorigenesis, at least in part via targeting the tumor suppressor pten. this microrna may serve as a potential diagnostic marker and therapeutic target for breast cancer.
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the mir-302-367 cluster is specifically expressed in human embryonic stem cells and has been shown to convert human somatic cells into induced pluripotent stem cells. here, we investigated the role of the mir-302-367 cluster in cervical carcinoma. the cluster was not endogenously expressed in cervical cancer cells, and its ectopic expression did not reprogram the cervical cancer cells to an embryonic stem cell-like state. however, ectopic expression of the mir-302-367 cluster in hela and siha cervical cancer cells inhibited cell proliferation and tumor formation by blocking the g1/s cell cycle transition. we identified a new cell cycle regulatory pathway in which the mir-302-367 cluster directly down-regulated both cyclin d1 and akt1 and indirectly up-regulated p27(kip1) and p21(cip1), leading to the suppression of cervical cancer cell proliferation. our findings suggest that the mir-302-367 cluster may be used as a therapeutic reagent for the treatment of cervical carcinoma.
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morphogenesis is crucial to initiate physiological development and tumor invasion. here we show that a microrna controls zonation morphogenesis by targeting hyaluronan receptor cd44. we have developed a novel system to study microrna functions by generating constructs expressing pre-mirnas and mature mirnas. using this system, we have demonstrated that expression of mir-328 reduced cell adhesion, aggregation, and migration, and regulated formation of capillary structure. protein analysis indicated that mir-328 repressed cd44 expression. activities of luciferase constructs harboring the target site in cd44, but not the one containing mutation, were repressed by mir-328. zonation morphogenesis appeared in cells transfected by mir-328: mir-328-transfected cells were present on the surface of zonating structures while the control cells stayed in the middle. mir-328-mediated cd44 actions was validated by anti-cd44 antibody, hyaluronidase, cd44 sirna, and cd44 expression constructs. in vivo experiments showed that cd44-silencing cells appeared as layers on the surfaces of nodules or zonating structures. immuno-histochemistry also exhibited cd44-negative cells on the surface layers of normal rat livers and the internal zones of portal veins. our results demonstrate that mir-328 targets cd44, which is essential in regulating zonation morphogenesis: silencing of cd44 expression is essential in sealing the zonation structures to facilitate their extension and to inhibit complex expansion.
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given that mir-124 is preferentially expressed in differentiating and mature neurons and external granule cells of cerebellum are thought to be cells-of-origins of medulloblastomas, we investigated if mir-124 played a role in the development of medulloblastomas. quantitative expression analysis of 29 medulloblastomas demonstrated significant down-regulation of mir-124 in 21 (72%) tumors by at least 2-fold, with 11 of them exhibiting greater than 10-fold reduced level compared to normal cerebella (p < .01). ectopic expression of mir-124 in medulloblastoma cell lines, ons-76 and daoy, inhibited cell proliferation. using computational and expression analyses, solute carrier family 16, member 1 (slc16a1) was identified as a candidate target of mir-124. transfection of mir-124 resulted in down-regulation of slc16a1 at both transcript and protein levels. reporter assay with 3' untranslated region of slc16a1 cloned downstream of the luciferase gene showed reduced luciferase activity in the presence of mir-124, providing strong evidence that mir-124 is a direct regulator of slc16a1. expression analysis further revealed that slc16a1 transcript was elevated in 26 (90%) of 29 tumors examined. knockdown of slc16a1 by sirna induced cell death in medulloblastoma cells. slc16a1 functions to efflux lactic acid during aerobic glycolysis. we speculated that inhibition of slc16a1 function resulted in a decrease of intracellular ph to a lethal level. in conclusion, our study demonstrates that mir-124 deregulation is common in medulloblastomas, and restoration of its function inhibits cell proliferation, suggesting that mir-124 may act as a growth suppressor. our findings also raise the possibility that the mir-124/slc16a1 pathway may represent a novel therapeutic target for treatment of malignant medulloblastomas.
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relatively little is known about the evolutionary histories of most classes of non-protein coding rnas. here we consider y rnas, a relatively rarely studied group of related pol-iii transcripts. a single cluster of functional genes is preserved throughout tetrapod evolution, which however exhibits clade-specific tandem duplications, gene-losses, and rearrangements.
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spaced synaptic depolarization induces rapid axon terminal growth and the formation of new synaptic boutons at the drosophila larval neuromuscular junction (nmj). here, we identify a novel presynaptic function for the calcium/calmodulin-dependent kinase ii (camkii) protein in the control of activity-dependent synaptic growth. consistent with this function, we find that both total and phosphorylated camkii (p-camkii) are enriched in axon terminals. interestingly, p-camkii appears to be enriched at the presynaptic axon terminal membrane. moreover, levels of total camkii protein within presynaptic boutons globally increase within one hour following stimulation. these effects correlate with the activity-dependent formation of new presynaptic boutons. the increase in presynaptic camkii levels is inhibited by treatment with cyclohexamide suggesting a protein-synthesis dependent mechanism. we have previously found that acute spaced stimulation rapidly downregulates levels of neuronal micrornas (mirnas) that are required for the control of activity-dependent axon terminal growth at this synapse. the rapid activity-dependent accumulation of camkii protein within axon terminals is inhibited by overexpression of activity-regulated mir-289 in motor neurons. experiments in vitro using a camkii translational reporter show that mir-289 can directly repress the translation of camkii via a sequence motif found within the camkii 3' untranslated region (utr). collectively, our studies support the idea that presynaptic camkii acts downstream of synaptic stimulation and the mirna pathway to control rapid activity-dependent changes in synapse structure.
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micrornas (mirnas) are thought to have an important role in tumor metastasis by regulating diverse cellular pathways. here, we describe the function and regulation network of mir-206 in gastric cancer (gc) metastasis. mir-206 expression was downregulated in gc cells especially in high metastatic potential cells and was also significantly decreased in metastatic lesions compared with their corresponding primary tumor samples. both gain- and loss-of-function studies confirmed that mir-206 significantly suppressed gc cell invasion and metastasis both in vitro and in vivo. mechanistically, paired box gene 3 (pax3) was identified as a functional target of mir-206 in gc cells. mir-206 inhibited gc metastasis by negatively regulating expression of pax3. in addition, pax3 expression was markedly higher in gc tissues than in adjacent non-cancerous tissues. gc patients with positive pax3 expression had shorter overall survival times. transwell assays and in vivo metastasis assays demonstrated that overexpression of pax3 significantly promoted the invasiveness and pulmonary metastasis of gc cells. on the other hand, downregulation of pax3 markedly reduced cell metastatic potential. mechanistic investigations indicated that prometastasis function of pax3 was mediated by upregulating downstream target met. moreover, we found that levels of pax3 and met were positively correlated in matched human gc specimens, and their coexpression was associated with poor prognoses. in conclusion, our results reveal that mir-206-pax3-met signaling is critical to gc metastasis. targeting the pathway described here may open new therapeutic prospects to restrict the metastatic potential of gc.
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micrornas (mirnas) are short noncoding rna molecules, which posttranscriptionally regulate genes expression and play crucial roles in diverse biological processes, such as development, differentiation, apoptosis and proliferation. here, we investigated the possible role of mirnas in the development of multidrug resistance (mdr) in human gastric and lung cancer cell lines. we found that mir-181b was downregulated in both multidrug-resistant human gastric cancer cell line sgc7901/vincristine (vcr) and multidrug-resistant human lung cancer cell line a549/cisplatin (cddp), and the downregulation of mir-181b in sgc7901/vcr and a549/cddp cells was concurrent with the upregulation of bcl2 protein, compared with the parental sgc7901 and a549 cell lines, respectively. in vitro drug sensitivity assay demonstrated that overexpression of mir-181b sensitized sgc7901/vcr and a549/cddp cells to anticancer drugs, respectively. the luciferase activity of a bcl2 3'-untranslated region-based reporter construct in sgc7901/vcr and a549/cddp cells suggests that a new target site in the 3'utr of bcl2 of the mature mir-181s (mir-181a, mir-181b, mir-181c and mir-181d) was found. enforced mir-181b expression reduced bcl2 protein level and sensitized sgc7901/vcr and a549/cddp cells to vcr-induced and cddp-induced apoptosis, respectively. taken together, our findings suggest that mir-181b could play a role in the development of mdr in both gastric and lung cancer cell lines, at least in part, by modulation of apoptosis via targeting bcl2.
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hepatic scavenger receptor class b type i (sr-bi) plays an important role in selective high-density lipoprotein cholesterol (hdl-c) uptake, which is a pivotal step of reverse cholesterol transport. in this study, the potential involvement of micrornas (mirnas) in posttranscriptional regulation of hepatic sr-bi and selective hdl-c uptake was investigated. the level of sr-bi expression was repressed by mirna 185 (mir-185), mir-96, and mir-223, while the uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (dii)-hdl was decreased by 31.9% (p < 0.001), 23.9% (p < 0.05), and 15.4% (p < 0.05), respectively, in hepg2 cells. the inhibition of these mirnas by their anti-mirnas had opposite effects in these hepatic cells. the critical effect of mir-185 was further validated by the loss of regulation in constructs with mutated mir-185 target sites. in addition, these mirnas directly targeted the 3' untranslated region (utr) of sr-bi with a coordinated effect. interestingly, the decrease of mir-96 and mir-185 coincided with the increase of sr-bi in the livers of apoe ko mice on a high-fat diet. these data suggest that mir-185, mir-96, and mir-223 may repress selective hdl-c uptake through the inhibition of sr-bi in human hepatic cells, implying a novel mode of regulation of hepatic sr-bi and an important role of mirnas in modulating cholesterol metabolism.
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ribonuclease p (rnase p) is a ribonucleoprotein enzyme that cleaves precursor trna transcripts to give mature 5' ends. rnase p in eubacteria has a large, catalytic rna subunit and a small protein subunit that are required for precursor trna cleavage in vivo. although the eukaryotic holoenzymes have similar, large rna subunits, previous work in a number of systems has suggested that the eukaryotic enzymes require a greater protein content. we have purified the saccharomyces cerevisiae nuclear rnase p to apparent homogeneity, allowing the first comprehensive analysis of an unexpectedly complex subunit composition. peptide sequencing by ion trap mass spectrometry identifies nine proteins that copurify with the nuclear rnase p rna subunit, totaling 20-fold more protein than in the bacterial enzyme. all of these proteins are encoded by genes essential for rnase p activity and for cell viability. previous genetic studies suggested that four proteins might be subunits of both rnase p and rnase mrp, the related rrna processing enzyme. we demonstrate that all four of these proteins, pop1p, pop3p, pop4p, and rpp1p, are integral subunits of rnase p. in addition, four of the five newly identified protein subunits, pop5p, pop6p, pop7p, and pop8p, also appear to be shared between rnase p and rnase mrp. only one polypeptide, rpr2p, is unique to the rnase p holoenzyme by genetic depletion and immunoprecipitation studies. the large increase in the number of protein subunits over eubacterial rnase p is consistent with an increase in functional complexity in eukaryotes. the degree of structural similarity between nuclear rnase p and rnase mrp suggests that some aspects of their functions in pre-trna and pre-rrna processing pathways might overlap or be coordinated.
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animal microrna (mirna) target prediction is still a challenge, although many prediction programs have been exploited. mirnas exert their function through partially binding the messenger rnas (mrnas; likely at 3' untranslated regions ), which makes it possible to detect the mirna-mrna interactions in vitro by co-transfection of mirna and a luciferase reporter gene containing the target mrna fragment into mammalian cells under a dual-luciferase assay system. here, we constructed a human mirna expression library and used a dual-luciferase assay system to perform large-scale screens of interactions between mirnas and the 3'utrs of seven genes, which included more than 3,000 interactions with triplicate experiments for each interaction. the screening results showed that the 3'utr of one gene can be targeted by multiple mirnas. among the prediction algorithms, a bayesian phylogenetic mirna target identification algorithm and a support vector machine (svm) presented a relatively better performance (27% for eimmo and 24.7% for mirdb) against the average precision (17.3%) of the nine prediction programs used here. additionally, we noticed that a relatively high conservation level was shown at the mirna 3' end targeted regions, as well as the 5' end (seed region) binding sites.
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there has been a great expansion in the number of small regulatory rnas identified in bacteria. some of these small rnas repress the synthesis of potentially toxic proteins. generally the toxin proteins are hydrophobic and less than 60 amino acids in length, and the corresponding antitoxin small rna genes are antisense to the toxin genes or share long stretches of complementarity with the target mrnas. given their short length, only a limited number of these type i toxin-antitoxin loci have been identified, but it is predicted that many remain to be found. already their characterization has given insights into regulation by small rnas, has suggested functions for the small toxic proteins at the cell membrane, and has led to practical applications for some of the type i toxin-antitoxin loci.
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pkr, an interferon (ifn)-inducible protein kinase activated by double-stranded rna, inhibits translation by phosphorylating the initiation factor eif2alpha chain. we show that human ifn-gamma mrna uses local activation of pkr in the cell to control its own translation yield. ifn-gamma mrna activates pkr through a pseudoknot in its 5' untranslated region. mutations that impair pseudoknot stability reduce the ability to activate pkr and strongly increase the translation efficiency of ifn-gamma mrna. nonphosphorylatable mutant eif2alpha, knockout of pkr and pkr inhibitors 2-aminopurine, transdominant-negative pkr, or vaccinia e3l correspondingly enhances translation of ifn-gamma mrna. the potential to form the pseudoknot is phylogenetically conserved. we propose that the rna pseudoknot acts to adjust translation of ifn-gamma mrna to the pkr level expressed in the cell.
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a 190-nucleotide (nt) packaging signal (ps) located in the 3' end of open reading frame 1b in the mouse hepatitis virus, a group iia coronavirus, was previously postulated to direct genome rna packaging. based on phylogenetic data and structure probing, we have identified a 95-nt hairpin within the 190-nt ps domain which is conserved in all group iia coronaviruses but not in the severe acute respiratory syndrome coronavirus (group iib), group i coronaviruses, or group iii coronaviruses. the hairpin is composed of six copies of a repeating structural subunit that consists of 2-nt bulges and 5-bp stems. we propose that repeating aa bulges are characteristic features of group iia pss.
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purpose microrna-21 (mirna-21) has proto-oncogenic properties, although no mirna-21-specific targets have been found in head and neck squamous cell carcinoma (hnscc). further study of mirna-21 and its specific targets is essential to understanding hnscc biology. experimental design mirna expression profiles of 10 hnsccs and 10 normal mucosa samples were investigated using a custom mirna microarray. thirteen hnsccs and five normal mucosa primary tissue specimens underwent mrna expression microarray analysis. to identify mirna-21 downstream targets, oral keratinocyte cells were subjected to microarray analysis after mirna-21 transient transfection. mirna and mrna expression were validated by reverse transcription quantitative polymerase chain reaction (rt-qpcr) in a separate cohort of 16 hnsccs and 15 normal mucosal samples. microarray and bioinformatics analyses were integrated to identify potential gene targets. in vitro assays looked at the function and interaction of mirna-21 and its specific gene targets. results mirna-21 was upregulated in hnsccs and stimulated cell growth. integrated analyses identified clusterin (clu) as a potential mirna-21 gene target. clu was downregulated after forced expression of mirna-21 in normal and hnscc cell lines. the activity of a luciferase construct containing the 3'-untranslated region (utr) of clu was repressed by the ectopic expression of mirna-21. clu was also downregulated in primary hnsccs and correlated with mirna-21 overexpression. clu variant 1 (clu-1) was the predominant splice variant in hnsccs and showed growth suppression function that was reversed by mirna-21 overexpression. conclusions clu is a specific, functional target of oncogenic mirna-21 in hnsccs. clu-1 isoform is the predominant growth-suppressive variant targeted by mirna-21.
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micrornas (mirnas) are essential components of gene regulation, but identification of mirna targets remains a major challenge. most target prediction and discovery relies on perfect complementarity of the mirna seed to the 3' untranslated region (utr). however, it is unclear to what extent mirnas target sites without seed matches. here, we performed a transcriptome-wide identification of the endogenous targets of a single mirna-mir-155-in a genetically controlled manner. we found that approximately 40% of mir-155-dependent argonaute binding occurs at sites without perfect seed matches. the majority of these noncanonical sites feature extensive complementarity to the mirna seed with one mismatch. these noncanonical sites confer regulation of gene expression, albeit less potently than canonical sites. thus, noncanonical mirna binding sites are widespread, often contain seed-like motifs, and can regulate gene expression, generating a continuum of targeting and regulation.
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the quest for non-coding rnas (ncrnas) in the last few years has revealed a surprisingly large number of small rnas belonging to previously known as well as entirely novel classes. computational and experimental approaches have uncovered new ncrnas in all kingdoms of life. in this work, we used a shotgun cloning approach to construct full-length cdna libraries of small rnas from the eukaryotic model organism dictyostelium discoideum. interestingly, two entirely novel classes of rnas were identified of which one is developmentally regulated. the rnas within each class share conserved 5'- and 3'-termini that can potentially form stem structures. rnas of both classes show predominantly cytoplasmic localization. in addition, based on conserved structure and/or sequence motifs, several of the identified ncrnas could be divided into classes known from other organisms, e.g. 18 small nucleolar rna candidates (17 box c/d, of which a few are developmentally regulated, and one box h/aca). two ncrnas showed a high degree of similarity to the small nuclear u2 rna and signal recognition particle rna (srp rna), respectively. furthermore, the majority of the regions upstream of the sequences encoding the isolated rnas share conserved motifs that may constitute new promoter elements.
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mir-185 expression has been associated with many cancers. however, the roles of mir-185 in human breast cancer remain elusive. here, we found that mir-185 expression was decreased in human breast cancer tissues compared with healthy tissue controls. up-regulation of mir-185 inhibited breast cancer cell proliferation and invasion and vice versa. mir-185 was shown to bind to the 3′-untranslated region (utr) of vascular endothelial growth factor a (vegfa), and a significant inverse correlation was found between mir-185 and vegfa. vegfa overexpression partially restored the inhibition of cell proliferation and invasion that was induced by mir-185, and vice versa. additionally, vegfa expression was found to be high in human breast cancer tissues. thus, mir-185-mediated vegfa targeting may be involved in breast cancer formation.
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7sk rna is a highly abundant noncoding rna in mammalian cells whose function in transcriptional regulation has only recently been elucidated. despite its highly conserved sequence throughout vertebrates, all attempts to discover 7sk rna homologues in invertebrate species have failed so far. here we report on a combined experimental and computational survey that succeeded in discovering 7sk rnas in most of the major deuterostome clades and in two protostome phyla: mollusks and annelids. despite major efforts, no candidates were found in any of the many available ecdysozoan genomes, however. the additional sequence data confirm the evolutionary conservation and hence functional importance of the previously described 3' and 5' stem-loop motifs, and provide evidence for a third, structurally well-conserved domain.
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micrornas (mirnas) play critical roles in retinoblastoma (rb) initiation and progression, aberrant expression of mir-145 had been frequently reported in cancer studies. however, the role and mechanism of its function in rb is still unclear. in this study, our data showed that mir-145 was downregulated in rb tissues and cell lines. overexpression of mir-145 suppressed rb cell proliferation, migration and invasion in vitro. adam19 was identified as a direct target of mir-145. silencing of adam19 significantly inhibited rb cell proliferation, migration and invasion. in addition, a reverse correlation between mir-145 and adam19 expression was noted in rb tissues. taken together, these findings suggested that mir-145 functions as a tumor suppressor in rb by directly targeting adam19. mir-145 could be an anticancer therapeutic target for rb patients.
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hodgkin lymphoma (hl) is derived from preapoptotic germinal center b cells, although a general loss of b cell phenotype is noted. using quantitative reverse transcription-polymerase chain reaction and mirna microarray, we determined the microrna (mirna) profile of hl and compared this with the profile of a panel of b-cell non-hodgkin lymphomas. the two methods showed a strong correlation for the detection of mirna expression levels. the hl-specific mirna included mir-17-92 cluster members, mir-16, mir-21, mir-24, and mir-155. using a large panel of cell lines, we found differential expression between hl and other b-cell lymphoma-derived cell lines for 27 mirna. a significant down-regulation in hl compared to non-hodgkin lymphoma was observed only for mir-150. next, we performed target gene validation of predicted target genes for mir-155, which is highly expressed in hl and is differentially expressed between hl and burkitt lymphoma. using luciferase reporter assays, we validated 11 predicted mir-155 target genes in three different hl cell lines. we demonstrated that agtr1, fgf7, znf537, zic3, and ikbke are true mir-155 target genes in hl.
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gata-4 is an important transcription factor involved in several developmental processes of the heart, such as cardiac myocyte proliferation, differentiation and survival. the precise mechanisms underlying the regulation of gata-4 remain unclear, this is especially true for the mechanisms that mediate the post-transcriptional regulation of gata-4. here, we demonstrate that mir-200b, a member of the mir-200 family, is a critical regulator of gata-4. overexpression of mir-200b leads to the downregulation of gata-4 mrna and a decrease in gata-4 protein levels. moreover, mir-200b not only inhibits cell growth and differentiation but also reverses the growth response mediated by gata-4, whereas depletion of mir-200b leads to a slight reversal of the anti-growth response achieved by knocking down endogenous gata-4. more importantly, the cell cycle-associated gene cyclin d1, which is a downstream target of gata-4, is also regulated by mir-200b. thus, mir-200b targets gata-4 to downregulate the expression of cyclin d1 and myosin heavy chain (mhc), thereby regulating cell growth and differentiation.
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how mirnas recognize their target sites is a puzzle that many experimental and computational studies aimed to solve. several features, such as perfect pairing of the mirna seed, additional pairing in the 3' region of the mirna, relative position in the 3' utr, and the a/u content of the environment of the putative site, have been found to be relevant. here we have used a large number of previously published data sets to assess the power that various sequence and structure features have in distinguishing between putative sites that do and those that do not appear to be functional. we found that although different data sets give widely different answers when it comes to ranking the relative importance of these features, the sites inferred from most transcriptomics experiments, as well as from comparative genomics, appear similar at this level. this suggests that mirna target sites have been selected in evolution on their ability to trigger mrna degradation. to understand at what step in the mirna-induced response individual features play a role, we transfected human hek293 cells with mirnas and analyzed the association of argonaute/eif2c-mirna complexes with target mrnas and the degradation of these messages. we found that structural features of the target site are only important for argonaute/eif2c binding, while sequence features such as the a/u content of the 3' utr are important for mrna degradation.
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aberrant expression of dna polymerase beta, a key enzyme involved in base excision repair, leads to genetic instability and carcinogenesis. pol beta expression has been previously shown to be regulated at the level of transcription, but there is also evidence of post-transcriptional regulation, since rat transcripts undergo alternative polyadenylation, and the resulting 3'utr contain at least one regulatory element. data presented here indicate that rna of the short 3'utr folds to form a strong secondary structure (hairpin). its regulatory role was established utilizing a luciferase-based reporter system. further studies led to the identification of a protein factor, which binds to this element-the anti-apoptotic, cytoskeleton-related protein hax-1. the results of in vitro binding analysis indicate that the formation of the rna-protein complex is significantly impaired by disruption of the hairpin motif. we demonstrate that hax-1 binds to pol beta mrna exclusively in the form of a dimer. biochemical analysis revealed the presence of hax-1 in mitochondria, but also in the nuclear matrix, which, along with its transcript-binding properties, suggests that hax-1 plays a role in post-transcriptional regulation of expression of pol beta.
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the currently available yeast mitochondrial dna (mtdna) sequence is incomplete, contains many errors and is derived from several polymorphic strains. here, we report that the mtdna sequence of the strain used for nuclear genome sequencing assembles into a circular map of 85,779 bp which includes 10 kb of new sequence. we give a list of seven small hypothetical open reading frames (orfs). hot spots of point mutations are found in exons near the insertion sites of optional mobile group i intron-related sequences. our data suggest that shuffling of mobile elements plays an important role in the remodelling of the yeast mitochondrial genome.
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polycomb group (pcg) proteins exert essential functions in the most disparate biological processes. the contribution of pcg proteins to cell commitment and differentiation relates to their ability to repress transcription of developmental regulators in embryonic stem (es) cells and in committed cell lineages, including skeletal muscle cells (smc). pcg proteins are preferentially removed from transcribed regions, but the underlying mechanisms remain unclear. here, pcg proteins are found to occupy and repress transcription from an intronic region containing the microrna mir-214 in undifferentiated smc. differentiation coincides with pcg disengagement, recruitment of the developmental regulators myod and myogenin, and activation of mir-214 transcription. once transcribed, mir-214 negatively feeds back on pcg by targeting the ezh2 3'utr, the catalytic subunit of the prc2 complex. mir-214-mediated ezh2 protein reduction accelerates smc differentiation and promotes unscheduled transcription of developmental regulators in es cells. thus, mir-214 and ezh2 establish a regulatory loop controlling pcg-dependent gene expression during differentiation.
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previously, two riboswitch classes have been identified that sense and respond to the hypermodified nucleobase called prequeuosine1 (preq1). the enormous expansion of available genomic dna sequence data creates new opportunities to identify additional representatives of the known riboswitch classes and to discover novel classes. we conducted bioinformatics searches on microbial genomic dna data sets to discover numerous additional examples belonging to the two previously known riboswitch classes for preq1 (classes preq1-i and preq1-ii), including some structural variants that further restrict ligand specificity. additionally, we discovered a third preq1-binding riboswitch class (preq1-iii) that is structurally distinct from previously known classes. these findings demonstrate that numerous organisms monitor the concentrations of this modified nucleobase by exploiting one or more riboswitch classes for this widespread compound.
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5s rrna extends from the central protuberance of the large ribosomal subunit, through the a-site finger, and down to the gtpase-associated center. here, we present a structure-function analysis of seven 5s rrna alleles which are sufficient for viability in the yeast saccharomyces cerevisiae when expressed in the absence of wild-type 5s rrnas, and extend this analysis using a large bank of mutant alleles that show semi-dominant phenotypes in the presence of wild-type 5s rrna. this analysis supports the hypothesis that 5s rrna serves to link together several different functional centers of the ribosome. data are also presented which suggest that in eukaryotic genomes selection has favored the maintenance of multiple alleles of 5s rrna, and that these may provide cells with a mechanism to post-transcriptionally regulate gene expression.
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recent studies suggest that microrna (mirna) plays important roles in the control of immune response and tolerance. we previously found that the expression level of antimicrobial peptide gene drosomycin (drs) is decreased in mir-964 overexpressing flies. here, we further verified that mir-964 deficiency leads to hyper-activation of drs. in addition, we employed three widely-used bioinformatic algorithms to screen potential mir-964 targets. finally, we identified that mir-964 modulates toll signaling pathway, at least in part, by repressing the expression of drs. taken together, our study identifies mir-964 as a modulator of toll signaling and enriches the repertoire of immune-modulating mirnas in drosophila.
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micrornas (mirna) regulate complex patterns of gene expression, and the relevance of altered mirna expression to ovarian cancer remains to be elucidated. by comprehensively profiling expression of mirnas and mrnas in serous ovarian tumors and cell lines and normal ovarian surface epithelium, we identified hundreds of potential mirna-mrna targeting associations underlying cancer. functional overexpression of mir-31, the most underexpressed mirna in serous ovarian cancer, repressed predicted mir-31 gene targets including the cell cycle regulator e2f2. mir31 and cdkn2a, which encode p14(arf) and p16(ink4a), are located at 9p21.3, a genomic region commonly deleted in ovarian and other cancers. p14(arf) promotes p53 activity, and e2f2 overexpression in p53 wild-type cells normally leads via p14(arf) to an induction of p53-dependent apoptosis. in a number of serous cancer cell lines with a dysfunctional p53 pathway (i.e., ovcar8, ovca433, and skov3), mir-31 overexpression inhibited proliferation and induced apoptosis; however, in other lines (i.e., hey and ovsayo) with functional p53, mir-31 had no effect. additionally, the osteosarcoma cell line u2os and the prostate cancer cell line pc3 (p14(arf)-deficient and p53-deficient, respectively) were also sensitive to mir-31. furthermore, mir-31 overexpression induced a global gene expression pattern in ovcar8 associated with better prognosis in tumors from patients with advanced stage serous ovarian cancer, potentially affecting many genes underlying disease progression. our findings reveal that loss of mir-31 is associated with defects in the p53 pathway and functions in serous ovarian cancer and other cancers, suggesting that patients with cancers deficient in p53 activity might benefit from therapeutic delivery of mir-31.
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the regulation of function of endothelial cell-cell junctions is fundamental in sustaining vascular integrity. the polycistronic microrna (mir) complexes containing mir-23a-27a-24-2, and 23b-27b-24-1 are predicted to target the majority of major endothelial junctional proteins. we focus on mir-23a and mir-23b, and investigate the functional effects of these mirs on junctions. while mir-23a and 23b only differ by 1 nucleotide (g19) outside the seed region and thus are predicted to have the same targets, they function differently with mir-23a inhibiting permeability and mir-23b inhibiting angiogenesis. both mirs target the junctional attractive molecule (tight junction protein 2) zo-2 and the repulsive molecule junctional adhesion molecule c (jam-c), although the inhibition of jam-c by mir-23a is more profound than by mir-23b. the difference in potency is attributable to differences at g19 since a mutation of the t17, the g19 binding site of mir-23b in the 3'utr of jam-c restores identity. we also show that the pattern of expression of mir-23a and mir-23b and their targets are different. thus, the paralogues mir-23a and mir-23b can have profoundly different effects on endothelial cell function due at least partially to selective effects on target proteins and differences in expression patterns of the mirs. this work exposes a hitherto unappreciated complexity in therapeutically targeting mirs.
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bioinformatics searches of eubacterial genomes have yielded many riboswitch candidates where the identity of the ligand is not immediately obvious on examination of associated genes. one of these motifs is found exclusively in the family streptococcaceae within the 5' untranslated regions (utrs) of genes encoding the hypothetical membrane protein classified as cog4708 or duf988. while the function of this protein class is unproven, a riboswitch binding the queuosine biosynthetic intermediate pre-queuosine(1) (preq(1)) has been identified in the 5' utr of homologous genes in many firmicute species of bacteria outside of streptococcaceae. here we show that a representative of the cog4708 rna motif from streptococcus pneumoniae r6 also binds preq(1). furthermore, representatives of this rna have structural and molecular recognition characteristics that are distinct from those of the previously described preq(1) riboswitch class. preq(1) is the second metabolite for which two or more distinct classes of natural aptamers exist, indicating that natural aptamers utilizing different structures to bind the same metabolite may be more common than is currently known. additionally, the association of preq(1) binding rnas with most genes encoding proteins classified as cog4708 strongly suggests that these proteins function as transporters for preq(1) or another queuosine biosynthetic intermediate.
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many or all of the sites of pseudouridine (psi) formation in eukaryotic rrna are selected by site-specific base-pairing with members of the box h + aca class of small nucleolar rnas (snornas). database searches previously identified strong homology between the rat nucleolar protein nap57p, its yeast homolog cbf5p, and the escherichia coli psi synthase trub/p35. we therefore tested whether cbf5p is required for synthesis of psi in the yeast rrna. after genetic depletion of cbf5p, formation of psi in the pre-rrna is dramatically inhibited, resulting in accumulation of the unmodified rrna. protein a-tagged cbf5p coprecipitates all tested members of the box h + aca snornas but not box c + d snornas or other rna species. genetic depletion of cbf5p leads to depletion of all box h + aca snornas. these include snr30, which is required for pre-rrna processing. depletion of cbf5p also results in a pre-rrna processing defect similar to that seen on depletion of snr30. we conclude that cbf5p is likely to be the rrna psi synthase and is an integral component of the box h + aca class of snornps, which function to target the enzyme to its site of action.
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mir-7 (microrna-7) has been characterized as a tumour suppressor in several human cancers. it targets a number of proto-oncogenes that contribute to cell proliferation and survival. however, the mechanism(s) by which mir-7 suppresses tumorigenesis in tscc (tongue squamous cell carcinoma) is unknown. the present bioinformatics analysis revealed that igf1r (insulin-like growth factor 1 receptor) mrna is a potential target for mir-7. ectopic transfection of mir-7 led to a significant reduction in igf1r at both the mrna and protein levels in tscc cells. knockdown of mir-7 in tscc cells enhanced igf1r expression. direct targeting of mir-7 to three candidate binding sequences located in the 3'-untranslated region of igf1r mrna was confirmed using luciferase-reporter-gene assays. the mir-7-mediated down-regulation of igf1r expression attenuated the igf1 (insulin-like growth factor 1)-induced activation of akt (protein kinase b) in tscc cell lines, which in turn resulted in a reduction in cell proliferation and cell-cycle arrest, and an enhanced apoptotic rate. taken together, the present results demonstrated that mir-7 regulates the igf1r/akt signalling pathway by post-transcriptional regulation of igf1r. our results indicate that mir-7 plays an important role in tscc and may serve as a novel therapeutic target for tscc patients.
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sensorineural hearing loss (snhl) is the most common cause of hearing impairment. one of the essential steps to prevent progressive hearing loss is to protect spiral ganglion neurons (sgns) from ongoing degeneration. micrornas and tmprss3 (transmembrane protease, serine 3) have been reported to be involved in development of sgns and genesis of snhl. the aim of this study was to investigate the role of mir-204 and tmprss3 in sgns. effect of mir-204 on cell viability of sgns was first examined using mtt (3--2,5 diphenyl tetrazolium bromide) assay. expression of tmprss3 in sgns with or without addition of mir-204 was assessed by real-time pcr and western blot further. a luciferase reporter activity assay was conducted to confirm target association between mir-204 and 3'-utr of tmprss3. finally, role of tmprss3 on cell viability of sgns was evaluated by transfection of tmprss3 sirna. cell viability of sgns was suppressed by mir-204 in a concentration-dependent manner. overexpression of mir-204 reduced expression of tmprss3 in sgns at both mrna and protein levels. binding to the 3'-utr of tmprss3 by mir-204 was identified by luciferase assay. knockdown of tmprss3 by sirna significantly inhibits cell viability of sgns. mir-204 could be a potential therapeutic target in sensorineural hearing loss.
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streptomyces are predominantly soil-dwelling bacteria that are best known for their multicellular life cycle and their prodigious metabolic capabilities. they are also renowned for their regulatory capacity and flexibility, with each species encoding >60 sigma factors, a multitude of transcription factors, and an increasing number of small regulatory rnas. here, we describe our characterization of a conserved small rna (srna), scr4677. in the model species streptomyces coelicolor, this srna is located in the intergenic region separating sco4677 (an anti-sigma factor-encoding gene) and sco4676 (a putative regulatory protein-encoding gene), close to the sco4676 translation start site in an antisense orientation. there appears to be considerable genetic interplay between these different gene products, with wild type expression of scr4677 requiring function of the anti-sigma factor sco4677, and scr4677 in turn influencing the abundance of sco4676-associated transcripts. the scr4677-mediated effects were independent of rnase iii (a double stranded rna-specific nuclease), with rnase iii having an unexpectedly positive influence on the level of sco4676-associated transcripts. we have shown that both sco4676 and sco4677 affect the production of the blue-pigmented antibiotic actinorhodin under specific growth conditions, and that this activity appears to be independent of scr4677.
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mirnas can have pleiotropic effects by targeting multiple genes belonging to diverse signalling networks. alternatively, mirnas can enhance the potency of their cellular effects by targeting multiple genes within the same genetic pathway. previously, we and others have demonstrated that mir-335 is a potent suppressor of tumour cell migration, invasion and metastasis, in part by targeting several genes involved in these cellular processes, including rock1, mapk1, lrg1, sp1 and sox4. here, we demonstrate that direct targeting of multiple members of the formin family of actin nucleators contributes to the inhibitory effects of mir-335 in neuroblastoma cells. we demonstrate that mir-335 regulates the expression of at least five formin family members and validate three family members, fmnl3, fmn2 and daam2, as direct targets of mir-335. the contribution of the formin family genes to cancer progression and metastasis has recently begun to emerge and here we demonstrate for the first time the ability of fmn2 and daam2 to regulate tumour cell migration and invasion, using sirna-mediated inhibition of each of these formin genes. finally, we demonstrate that the formin genes, in particular fmnl3, are responsible for the protrusion of actin-rich filopodia structures that contribute to the enhanced migratory and invasive potential associated with reduced expression of mir-335. thus, direct targeting of the formin family contributes to the metastasis suppressing abilities of mir-335 by providing a direct regulatory link to the actin assembly machinery of the cell. we conclude that mir-335 is a master regulator of tumour cell migration and invasion by directly targeting a plethora of genes that effectively control cell migratory processes.
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aim microangiopathy due to endothelial dysfunction is a major contributing factor to the development of diabetes-induced cardiovascular disease (cvd). dysregulation of endothelial-specific micrornas (mirs) is correlated with impaired angiogenesis and cell survival. we investigated the profile of two angiomirs, mir-126, and mir-132, in the plasma of type 2 diabetic individuals without any known history of cvd as well as in the cardiac tissues collected from diabetics undergoing cardiac surgery. methods and results the presence of diabetes alone significantly decreased both angiomirs in the plasma and the myocardium. the down-regulation of angiomirs was also associated with reduced capillaries and arterioles and increased endothelial cell apoptosis, the hallmark of microangiopathy. importantly, a time course study in a type 2 diabetic mouse model confirmed that the down-regulation of angiomirs preceded endothelial apoptosis as well as alterations in the density of the microvasculature. finally, therapeutic overexpression of both angiomirs in diabetic aortic rings and human umbilical vein endothelial cells exposed to high glucose (hg) abrogated the deleterious effects of diabetes and hg on cell survival and proliferation and restored their angiogenic potential. conclusions these novel findings demonstrate that the down-regulation of angiomirs is a major underlying mechanism for the development of microangiopathy in diabetic hearts. therefore, therapeutic restoration of angiomirs could become a potential approach to combat the cardiovascular complications of diabetes.
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toll-like receptors (tlrs) are important pathogen recognition molecules and are key to epithelial immune responses to microbial infection. however, the molecular mechanisms that regulate tlr expression in epithelia are obscure. micro-rnas play important roles in a wide range of biological events through post-transcriptional suppression of target mrnas. here we report that human biliary epithelial cells (cholangiocytes) express let-7 family members, micro-rnas with complementarity to tlr4 mrna. we found that let-7 regulates tlr4 expression via post-transcriptional suppression in cultured human cholangiocytes. infection of cultured human cholangiocytes with cryptosporidium parvum, a parasite that causes intestinal and biliary disease, results in decreased expression of primary let-7i and mature let-7 in a myd88/nf-kappab-dependent manner. the decreased let-7 expression is associated with c. parvum-induced up-regulation of tlr4 in infected cells. moreover, experimentally induced suppression or forced expression of let-7i causes reciprocal alterations in c. parvum-induced tlr4 protein expression, and consequently, infection dynamics of c. parvum in vitro. these results indicate that let-7i regulates tlr4 expression in cholangiocytes and contributes to epithelial immune responses against c. parvum infection. furthermore, the data raise the possibility that micro-rna-mediated post-transcriptional pathways may be critical to host-cell regulatory responses to microbial infection in general.
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microrna 21 (mir-21) has been implicated in various aspects of carcinogenesis. however, its function and molecular mechanism in cervical squamous carcinoma have not been studied. using taqman quantitative real-time pcr and northern blot, we confirmed that mir-21 is significantly overexpressed in human cervical squamous cancer tissues and cell lines. remarkably, we showed that the level of mir-21 correlates with the tumor differentiation and nodal status by ish. furthermore, we demonstrated that mir-21 regulates proliferation, apoptosis, and migration of hpv16-positive cervical squamous cells. in order to identify candidate target genes for mir-21, we used gene expression profiling. by luciferase reporter assays, we confirmed that ccl20 is one of its target genes, which is related to the hpv16 e6 and e7 oncogenes. our results suggest that mir-21 may be involved in cervical squamous cell tumorigenesis.
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nidoviruses produce an extensive 3'-coterminal nested set of subgenomic mrnas, which are used to express their structural proteins. in addition, arterivirus and coronavirus mrnas contain a common 5' leader sequence, derived from the genomic 5' end. the joining of this leader sequence to different segments (mrna bodies) from the genomic 3'-proximal region presumably involves a unique mechanism of discontinuous minus-strand rna synthesis. key elements in this process are the so-called transcription-regulating sequences (trss), which determine a base-pairing interaction between sense and antisense viral rna that is essential for leader-to-body joining. to identify rna structures in the 5'-proximal region of the equine arteritis virus genome that may be involved in subgenomic mrna synthesis, a detailed secondary rna structure model was established using bioinformatics, phylogenetic analysis, and rna structure probing. according to this structure model, the leader trs is located in the loop of a prominent hairpin (leader trs hairpin; lth). the importance of the lth was supported by the results of a mutagenesis study using an eav molecular clone. besides evidence for a direct role of the lth in subgenomic rna synthesis, indications for a role of the lth region in genome replication and/or translation were obtained. similar lth structures could be predicted for the 5'-proximal region of all arterivirus genomes and, interestingly, also for most coronaviruses. thus, we postulate that the lth is a key structural element in the discontinuous subgenomic rna synthesis and is likely critical for leader trs function.
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micrornas (mirnas) are a class of 20-24 nt non-coding rnas that regulate gene expression primarily through post-transcriptional repression or mrna degradation in a sequence-specific manner. the roles of mirnas are just beginning to be understood, but the study of mirna function has been limited by poor understanding of the general principles of gene regulation by mirnas. here we used cne cells from a human nasopharyngeal carcinoma cell line as a cellular system to investigate mirna-directed regulation of vegf and other angiogenic factors under hypoxia, and to explore the principles of gene regulation by mirnas. through computational analysis, 96 mirnas were predicted as putative regulators of vegf. but when we analyzed the mirna expression profile of cne and four other vegf-expressing cell lines, we found that only some of these mirnas could be involved in vegf regulation, and that vegf may be regulated by different mirnas that were differentially chosen from 96 putative regulatory mirnas of vegf in different cells. some of these mirnas also co-regulate other angiogenic factors (differential regulation and co-regulation principle). we also found that vegf was regulated by multiple mirnas using different combinations, including both coordinate and competitive interactions. the coordinate principle states that mirnas with independent binding sites in a gene can produce coordinate action to increase the repressive effect of mirnas on this gene. by contrast, the competitive principle states when multiple mirnas compete with each other for a common binding site, or when a functional mirna competes with a false positive mirna for the same binding site, the repressive effects of mirnas may be decreased. through the competitive principle, false positive mirnas, which cannot directly repress gene expression, can sometimes play a role in mirna-mediated gene regulation. the competitive principle, differential regulation, multi-mirna binding sites, and false positive mirnas might be useful strategies in the avoidance of unwanted cross-action among genes targeted by mirnas with multiple targets.
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hedgehog (hh) pathway signaling is crucial for the maintenance of blood cell progenitors in the lymph gland hematopoietic organ present in drosophila third instar larvae. previous studies from our lab have likewise shown the importance of the mir-7 and bag of marbles (bam) genes in maintaining the progenitor state. thus, we sought to investigate a possible interaction between the hh pathway and mir-7/bam in the prohemocyte population within this hematopoietic tissue. gain of function mir-7 was able to rescue a blood cell progenitor depletion phenotype caused by patched (ptc) inhibition of hh pathway signaling in these cells. similarly, expression of a dominant/negative version of ptc was able to rescue the severe reduction of prohemocytes due to bam loss of function. furthermore, we demonstrated that suppressor of fused , another known inhibitor of hh signaling, likely serves as a translational repression target of the mir-7 mirna. our results suggest the mir-7/bam combination regulates the hh signaling network through repression of su(fu) to maintain hemocyte progenitors in the larval lymph gland.
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although there is abundant evidence that individual microrna (mirna) loci repress large cohorts of targets, large-scale knockout studies suggest that most mirnas are phenotypically dispensable. here, we identify a rare case of developmental cell specification that is highly dependent on mirna control of an individual target. we observe that binary cell fate choice in the drosophila melanogaster peripheral sensory organ lineage is controlled by the non-neuronally expressed mir-279/996 cluster, with a majority of notum sensory organs exhibiting transformation of sheath cells into ectopic neurons. the mir-279/996 defect phenocopies notch loss of function during the sheath-neuron cell fate decision, suggesting the mirnas facilitate notch signaling. consistent with this, mir-279/996 knockouts are strongly enhanced by notch heterozygosity, and activated nuclear notch is impaired in the mirna mutant. although hairless (h) is the canonical nuclear notch pathway inhibitor, and h heterozygotes exhibit bristle cell fate phenotypes reflecting gain-of-notch signaling, h/+ does not rescue mir-279/996 mutants. instead, we identify insensible (insb), another neural nuclear notch pathway inhibitor, as a critical direct mir-279/996 target. insb is posttranscriptionally restricted to neurons by these mirnas, and its heterozygosity strongly suppresses ectopic peripheral nervous system neurons in mir-279/996 mutants. thus, proper assembly of multicellular mechanosensory organs requires a double-negative circuit involving mirna-mediated suppression of a notch repressor to assign non-neuronal cell fate.
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notch (n) signaling is central to the self-renewal of neural stem cells (nscs) and other tissue stem cells. its deregulation compromises tissue homeostasis and contributes to tumorigenesis and other diseases. how n regulates stem cell behavior in health and disease is not well understood. here we show that n regulates bantam (ban) microrna to impact cell growth, a process key to nsc maintenance and particularly relied upon by tumor-forming cancer stem cells. notch signaling directly regulates ban expression at the transcriptional level, and ban in turn feedback regulates n activity through negative regulation of the notch inhibitor numb. this feedback regulatory mechanism helps maintain the robustness of n signaling activity and nsc fate. moreover, we show that a numb-myc axis mediates the effects of ban on nucleolar and cellular growth independently or downstream of n. our results highlight intricate transcriptional as well as translational control mechanisms and feedback regulation in the n signaling network, with important implications for nsc biology and cancer biology.
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lung cancer is the most common type of cancer-related death in developed countries. micrornas (mirnas) are small non-coding rnas, which regulates gene expression in cancer. recent studies demonstrate that the microrna-293-3p (mir-293-3p) may play as an oncogene or a tumor suppressor. however, its expression and roles in non-small cell lung cancer (nsclc) is not known. in this study, our purpose is to investigate the expression and roles of mir-296-3p in nsclc. the findings indicated that mir296-3p inhibited nsclc cell proliferation, enhance the drug resistance, and apoptosis. data of luciferase reporter assays demonstrated that the cx3cr1 gene was a direct regulator of tumorsuppressive mir296-3p. moreover, overexpressed cx3cr1 was confirmed in nsclc clinical specimens. inhibition of cx3cr1 could inhibit cancer cellular survival and increase chemotherapy sensitivity. there was a negative relationship between mir296-3p and cx3cr1 expression in nsclc tissues. our study elucidates that mir296-3p plays a suppressive role in nsclc by inhibiting cx3cr1 expression.
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objective abdominal aortic aneurysm (aaa) is characterized as a progressive dilation and degradation of the aortic wall, associated with activation of matrix metalloproteinases (mmps) and inflammation. emerging evidence indicates a role for micrornas (mirnas) in aaa pathogenesis, but it is unclear whether abdominal aortic endothelial mirnas play a role in the disease process. we aimed to identify mirnas in the abdominal aortic endothelium that play a critical role in aaa development. approach and results the mouse model of aaa induced by angiotensin ii infusion was used in this study. through a mirna array and validation study, we initially identified the murine-specific mir-712 and subsequently its human/murine homolog mir-205 as angiotensin ii-induced mirnas in the abdominal aortic endothelium in vivo and in vitro. mechanistically, mir-712 stimulated mmp activity in the aortic wall by directly targeting 2 mmp inhibitors: tissue inhibitor of metalloproteinase 3 (timp3) and reversion-inducing cysteine-rich protein with kazal motifs (reck). silencing of mir-712 and mir-205 by using anti-mir-712 and anti-mir-205, respectively, significantly decreased the aortic mmp activity and inflammation, preventing aaa development in angiotensin ii-infused apoe(-/-) mice. further, upregulation of 4 angiotensin ii-sensitive mirnas, mir-205, -21, -133b, and -378, identified in this murine study were confirmed in human aaa samples compared with nondiseased control. conclusions our results demonstrate that angiotensin ii-sensitive mir-712 and its human homolog mir-205 downregulate timp3 and reck, which in turn stimulate aortic mmp activity and inflammation, leading to aaa development. targeting these mirnas may be a novel therapeutic strategy to prevent aaa.
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cutaneous homeostasis and innate immunity is procured by a complex circuitry of intercellular cytokine signaling. micrornas are important posttranscriptional regulators of keratinocyte gene expression and assist in modulating the fine balance between cell proliferation and differentiation in skin. a characteristic microrna profile in inflammatory skin suggests putative functions of micrornas in perturbed cytokine production and signaling during chronic inflammatory skin conditions such as psoriasis. it remains unclear, however, why certain micrornas are aberrantly expressed during skin inflammation and if they serve pro- and/or anti-inflammatory functions. in this report, we focus on cytokine regulation by microrna-203 (mir-203), which is highly abundant in keratinocytes and upregulated in psoriatic lesions. by screening a panel of cytokines that are upregulated in psoriatic skin for regulation by mir-203, we identify the genes encoding the pro-inflammatory cytokines tnfα and il24 as direct targets of mir-203. studies of mir-203 overexpression, inhibition, and mutagenesis validate posttranscriptional regulation of tnfα and il24 by mir-203 in cell lines and primary keratinocytes. our findings suggest that mir-203 serves to fine-tune cytokine signaling and may dampen skin immune responses by repressing key pro-inflammatory cytokines.
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rna-based therapeutics are emerging as innovative options for cancer treatment, with micrornas being attractive targets for therapy development. we previously implicated microrna-642a-5p (mir-642a-5p) as a tumor suppressor in prostate cancer (pca), and here we characterize its mode of action, using 22rv1 pca cells. in an in vivo xenograft tumor model, mir-642a-5p induced a significant decrease in tumor growth, compared to negative control. using rna-sequencing, we identified gene targets of mir-642a-5p which were enriched for gene sets controlling cell cycle; downregulated genes included wilms tumor 1 gene (wt1), nuak1, rassf3 and skp2; and upregulated genes included igfbp3 and gps2. analysis of pca patient datasets showed a higher expression of wt1, nuak1, rassf3 and skp2; and a lower expression of gps2 and igfbp3 in pca tissue compared to non-malignant prostate tissue. we confirmed the prostatic oncogene wt1, as a direct target of mir-642a-5p, and treatment of 22rv1 and lncap pca cells with wt1 sirna or a small molecule inhibitor of wt1 reduced cell proliferation. taken together, these data provide insight into the molecular mechanisms by which mir-642a-5p acts as a tumor suppressor in pca, an effect partially mediated by regulating genes involved in cell cycle control; and restoration of mir-642-5p in pca could represent a novel therapeutic approach.
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co(2) sensation represents an interesting example of nervous system and behavioral evolutionary divergence. the underlying molecular mechanisms, however, are not understood. loss of microrna-279 in drosophila melanogaster leads to the formation of a co(2) sensory system partly similar to the one of mosquitoes. here, we show that a novel allele of the pleiotropic transcription factor prospero resembles the mir-279 phenotype. we use a combination of genetics and in vitro and in vivo analysis to demonstrate that pros participates in the regulation of mir-279 expression, and that reexpression of mir-279 rescues the pros co(2) neuron phenotype. we identify common target molecules of mir-279 and pros in bioinformatics analysis, and show that overexpression of the transcription factors nerfin-1 and escargot (esg) is sufficient to induce formation of co(2) neurons on maxillary palps. our results suggest that prospero restricts co(2) neuron formation indirectly via mir-279 and directly by repressing the shared target molecules, nerfin-1 and esg, during olfactory system development. given the important role of pros in differentiation of the nervous system, we anticipate that mir-mediated signal tuning represents a powerful method for olfactory sensory system diversification during evolution.
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spontaneous self-terminating atrial fibrillation (af) is one of the most common heart rhythm disorders, yet the regulatory molecular mechanisms underlying this syndrome are rather unclear. microrna (mirna) transcriptome and expression of candidate transcription factors (tfs) with potential roles in arrhythmogenesis, such as pitx2, tbx5, and myocardin (myocd), were analyzed by microarray, qrt-pcr, and western blotting in left atrial (la) samples from pigs with transitory af established by right atrial tachypacing. induced ectopic tachyarrhythmia caused rapid and substantial mirna remodeling associated with a marked downregulation of pitx2, tbx5, and myocd expression in atrial myocardium. the downregulation of pitx2, tbx5, and myocd was inversely correlated with upregulation of the corresponding targeting mirnas (mir-21, mir-10a/10b, and mir-1, resp.) in the la of paced animals. through in vitro transient transfections of hl-1 atrial myocytes, we further showed that upregulation of mir-21 did result in downregulation of pitx2 in cardiomyocyte background. the results suggest that immediate-early mirna remodeling coupled with deregulation of tf expression underlies the onset of af.
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background/aims the objective of this study was to investigate the potential role of il-17 in the development of nasopharyngeal carcinoma (npc) and to screen micrornas (mirnas) that potentially target il-17 in npc cells. methods blood was collected from npc patients and normal subjects, and plasma il-17 concentration was quantified by enzyme-linked immunosorbent assay. an immortalized normal human nasopharyngeal epithelial cell line, np69, was treated with or without human il-17 (15 ng/ml) for various times, and expression of il-1ß, il-6, il-12, and tnf-α mrna was assessed by real-time reverse transcription pcr. the candidate mirnas that potentially target il-17 were predicted by a bioinformatics strategy. the selected mir-135a mimic was transfected into primary npc cells, and cell proliferation was assessed by mtt assay. results the concentration of plasma il-17 was significantly higher in the npc patients (92.5 ± 7.3 pg/ml) than in the control subjects (56.8 ± 2.9 pg/ml). in response to il-17 treatment, the mrna expression of il-1ß and il-6 was significantly upregulated and reached a peak at 12 h, followed by a slight decrease at 24 h, while the mrna expression of il-12 and tnf-α was significantly upregulated at 12 h and remained high even at 48 h after exposure to il-17. moreover, mir-135a specifically targets il-17 and was dramatically downregulated in npc cells compared with np69 cells. transfection of exogenous mir-135a mimic resulted in significant suppression of il-17 secretion and subsequent inhibition of npc cell proliferation. conclusions blood il-17 was significantly higher in npc patients compared with normal subjects. expression of mir-135a in the cancer cells isolated from nasopharyngeal tumors was significantly lower than that in np69 cells, and suppression of il-17 by mir-135a mimic resulted in significant inhibition of npc cell proliferation. these findings suggested that downregulation of mir-135a may contribute to the development of npc via the mechanism of il-17 stimulation of proinflammatory cytokine expression.
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x inactivation is controlled by xist and its antisense gene, tsix, neither of which encodes a protein. xist is essential for x inactivation to occur in cis, and its differential expression is a key event in the initiation of x inactivation. xist and tsix are imprinted in the extraembryonic tissues of mouse embryos so that they are expressed from the paternal and maternal x, respectively, resulting in the preferential inactivation of the paternal x. targeted disruption of tsix causes ectopic expression of xist, suggesting that tsix negatively regulates xist in cis. however, the molecular mechanism of this antisense regulation remains unknown. here, we demonstrate that tsix transcriptionally silences xist in both embryonic and extraembryonic tissues of mouse embryos. moreover, we show that disruption of tsix impairs establishment of repressive epigenetic modifications and chromatin structure at the xist locus. we propose that tsix silences xist through modification of the chromatin structure.
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chronic infection with hepatotropic viruses is the main cause of chronic liver disease and cirrhosis worldwide. toll-like receptor 3 (tlr3) and toll-like receptor 7 (tlr7) are pathogen-recognition receptors that are expressed on innate immune cells. they recognize viral rna, which induces their activation, with a subsequent increase in type i interferon transcription. hepatitis c virus (hcv) infection inhibits the expression of tlr3 and tlr7; however, the mechanism by which this occurs is unclear. micrornas (mirnas) are small rnas that posttranscriptionally regulate gene expression. their aberrant expression is commonly correlated with disease status, as is the case with hcv infection. here, we found that mir-758 levels were increased in patients with hcv infection and were correlated with tlr3 and tlr7 expression levels in the patients with hcv infection, and bioinformatics analysis predicted that tlr3 and tlr7 are targets of mir-758. therefore, we postulate that hcv may increase the level of mir-758, which inhibits the expression of tlr3 and tlr7, resulting in a loss of antiviral effect. in order to test our hypothesis, we constructed an hcv core protein expression plasmid and used it to transfect liver cells. the results showed that hcv infection increased mir-758 levels and decreased tlr3/tlr7 expression. furthermore, using rt-pcr and luciferase reporter analysis, we found that mir-758 targets tlr3 and tlr7, with a subsequent decrease in ifnα and ifnβ production. in conclusion, our results highlight the upregulation of mir-758 expression by hcv as a novel mechanism contributing to downregulation of tlr3 and tlr7 in patients with hcv infection.
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akt2 is a crucial mediator in the tumorigenesis and thought to be an ideal target for the treatment of malignancies. increasing evidence has also shown that micrornas (mirnas) could regulate or be regulated by mrnas and they also might serve as therapeutic agencies or targets. mir-29s (mir-29a, mir-29b and mir-29c) had been approved that they decreased in gastric cancers (gc) by targeting ccnd2, mmp2 and p42.3. however, whether mir-29s would target akt2 have not been investigated in gc. here, we explored the relationship between mir-29s and akt2 and found that in gc cell lines (hgc-27 and mgc-803) and gc clinical samples the decreased levels of mir-29s accompanied by increased akt2 expression. introduction of mir-29s into gc cells resulted in decreased akt2 expression and decreased the ability of cancer cells invasion, so did the sirna-akt2. our studies revealed that mir-29s expression is downregulated in gc and they could repress the akt2 expression and the inactivation of akt and gsk3beta leading to inhibit the gc cells invasion. taken together, our findings suggested that akt2 may be one of the targets of mir29s in gastric cancer. by increasing the expression of mir-29s or decreasing akt2 expression may be promising in combating with gc.
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rationale viral myocarditis results from an adverse immune response to cardiotropic viruses, which causes irreversible myocyte destruction and heart failure in previously healthy people. the involvement of micrornas and their usefulness as therapeutic targets in this process are unknown. objective to identify micrornas involved in viral myocarditis pathogenesis and susceptibility. methods and results cardiac micrornas were profiled in both human myocarditis and in coxsackievirus b3-injected mice, comparing myocarditis-susceptible with nonsusceptible mouse strains longitudinally. microrna responses diverged depending on the susceptibility to myocarditis after viral infection in mice. microrna-155, -146b, and -21 were consistently and strongly upregulated during acute myocarditis in both humans and susceptible mice. we found that microrna-155 expression during myocarditis was localized primarily in infiltrating macrophages and t lymphocytes. inhibition of microrna-155 by a systemically delivered lna-anti-mir attenuated cardiac infiltration by monocyte-macrophages, decreased t lymphocyte activation, and reduced myocardial damage during acute myocarditis in mice. these changes were accompanied by the derepression of the direct microrna-155 target pu.1 in cardiac inflammatory cells. beyond the acute phase, microrna-155 inhibition reduced mortality and improved cardiac function during 7 weeks of follow-up. conclusions our data show that cardiac microrna dysregulation is a characteristic of both human and mouse viral myocarditis. the inflammatory microrna-155 is upregulated during acute myocarditis, contributes to the adverse inflammatory response to viral infection of the heart, and is a potential therapeutic target for viral myocarditis.
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background myocardial infarction leads to cardiac remodeling and development of heart failure. insufficient myocardial capillary density after myocardial infarction has been identified as a critical event in this process, although the underlying mechanisms of cardiac angiogenesis are mechanistically not well understood. methods and results here, we show that the small noncoding rna microrna-24 (mir-24) is enriched in cardiac endothelial cells and considerably upregulated after cardiac ischemia. mir-24 induces endothelial cell apoptosis, abolishes endothelial capillary network formation on matrigel, and inhibits cell sprouting from endothelial spheroids. these effects are mediated through targeting of the endothelium-enriched transcription factor gata2 and the p21-activated kinase pak4, which were identified by bioinformatic predictions and validated by luciferase gene reporter assays. respective downstream signaling cascades involving phosphorylated bad (bcl-xl/bcl-2-associated death promoter) and sirtuin1 were identified by transcriptome, protein arrays, and chromatin immunoprecipitation analyses. overexpression of mir-24 or silencing of its targets significantly impaired angiogenesis in zebrafish embryos. blocking of endothelial mir-24 limited myocardial infarct size of mice via prevention of endothelial apoptosis and enhancement of vascularity, which led to preserved cardiac function and survival. conclusions our findings indicate that mir-24 acts as a critical regulator of endothelial cell apoptosis and angiogenesis and is suitable for therapeutic intervention in the setting of ischemic heart disease.
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three elements are crucial for the programmed frameshifting in translation of dnax mrna: a shine-dalgarno (sd)-like sequence, a double-shift site, and a 3' structure. the conformation of the mrna containing these three elements was investigated using chemical and enzymatic probes. the probing data show that the structure is a specific stem-loop. the bottom half of the stem is more stable than the top half of the stem. the function of the stem-loop was further investigated by mutagenic analysis. reducing the stability of the bottom half of the stem strongly effects frameshifting levels, whereas similar changes in the top half are not as effective. stabilizing the top half of the stem gives increased frameshifting beyond the wt efficiency. the identity of the primary rna sequence in the stem-loop is unimportant, provided that the overall structure is maintained. the calculated stabilities of the variant stem-loop structures correlate with frameshifting efficiency. the sd-interaction and the stem-loop element act independently to increase frameshifting in dnax.
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msx1 is a key factor for the development of tooth and craniofacial skeleton and has been proposed to play a pivotal role in terminal cell differentiation. in this paper, we demonstrated the presence of an endogenous msx1 antisense rna (msx1-as rna) in mice, rats, and humans. in situ analysis revealed that this rna is expressed only in differentiated dental and bone cells with an inverse correlation with msx1 protein. these in vivo data and overexpression of msx1 sense and as rna in an odontoblastic cell line (mo6-g3) showed that the balance between the levels of the two msx1 rnas is related to the expression of msx1 protein. to analyze the impact of this balance in the msx-dlx homeoprotein pathway, we analyzed the effect of msx1, msx2, and dlx5 overexpression on proteins involved in skeletal differentiation. we showed that the msx1-as rna is involved in crosstalk between the msx-dlx pathways because its expression was abolished by dlx5. msx1 was shown to down-regulate a master gene of skeletal cells differentiation, cbfa1. all these data strongly suggest that the ratio between msx1 sense and antisense rnas is a very important factor in the control of skeletal terminal differentiation. finally, the initiation site for msx1-as rna transcription was located by primer extension in both mouse and human in an identical region, including a consensus tata box, suggesting an evolutionary conservation of the as rna-mediated regulation of msx1 gene expression.
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objective single nucleotide polymorphisms (snps) located at microrna (mirna) binding sites (mir-snps) can affect the expression of genes. this study aimed to identify the mir-snps associated with atherosclerosis and stroke. methods patients with ischemic stroke (n = 657) and stroke- and myocardial infarction-free volunteers (n = 1571) were enrolled. the carotid intima-media thickness (imt) was measured in the control participants. seventy-nine stroke susceptibility genes were initially selected and 13 genes were predicted to have mir-snps at their 3' untranslated regions (3'utr). the mirna arrays were used to further identify potential mir-snps. the mir-snp rs3735590 at the paraoxonase 1 (pon1) gene was finally selected and its associations with stroke and carotid imt were evaluated. the 3'utr reporter and snp functional assays were then performed to validate the results. results compared with cc genotype, patients with ct or tt genotype at rs3735590 had lower risk of ischemic stroke (or = 0.72, p = 0.036; or = 0.83, p = 0.077, respectively). among the healthy participants, the ct or tt genotype was associated with thinner imt in the internal carotid arteries in comparison with cc genotype (β = -0.76, p = 0.003; β = -0.022, p = 0.452, respectively). our findings suggested that the minor allele t had a protective effect on atherosclerosis. results from 3'utr reporter assays showed that pon1 is a direct target gene of mir-616. in plasmid constructs carrying the risk allele c at rs3735590, mir-616 inhibited the genetic expression of pon1. however, substitution of c by t at rs3735590 reduced the mir-616 binding affinity, leading to overexpression of the pon1 gene. conclusion our study is the first to show that the mir-snp at pon1 could affect genetic expression and is associated with an elevated risk for ischemic stroke and subclinical atherosclerosis.
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brca1, a breast and ovarian tumor suppressor, colocalizes with markers of the inactive x chromosome (xi) on xi in female somatic cells and associates with xist rna, as detected by chromatin immunoprecipitation. breast and ovarian carcinoma cells lacking brca1 show evidence of defects in xi chromatin structure. reconstitution of brca1-deficient cells with wt brca1 led to the appearance of focal xist rna staining without altering xist abundance. inhibiting brca1 synthesis in a suitable reporter line led to increased expression of an otherwise silenced xi-located gfp transgene. these observations suggest that loss of brca1 in female cells may lead to xi perturbation and destabilization of its silenced state.
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cyclin-dependent kinase inhibitor 1a (cdkn1a), also known as p21cip1/waf1, is a master downstream effector of tumor suppressors. in this study, we experimentally demonstrate through a high-throughput luciferase reporter screen that p21cip1/waf1 can be directly targeted by nearly 28 micrornas (mirnas). the results were further confirmed by a series of mutational analyses and luciferase reporter assays. these 28 mirnas can substantially inhibit p21cip1/waf1 expression, predominantly at translational level. many of these mirnas were upregulated in cancers and might serve as modulators of oncogenesis. furthermore, 8 of these 28 p21-regulating mirnas are located in the chromosome 19 mirna cluster, the largest mirna gene cluster in humans, and they can clearly promote cell proliferation and cell-cycle progression in choriocarcinoma cells. in conclusion, our screening strategy provides an alternative approach to uncovering mirna modulators of an individual mrna, and it has identified multiple mirnas that can suppress p21cip1/waf1 expression by directly targeting its 3' untranslated region.
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microrna-1 (mir-1) is preferentially expressed in cardiac muscles, and the expression has been demonstrated to be involved in cardiac development and cardiovascular diseases. here we report that mir-1 is closely related with ischemia/reperfusion injury in a rat model. the level of mir-1 is inversely correlated with bcl-2 protein expression in cardiomyocytes of the i/r rat model. in vitro, the level of mir-1 was dramatically increased in response to h(2)o(2). overexpression of mir-1 facilitated h(2)o(2)-induced apoptosis in cardiomyocytes. inhibition of mir-1 by antisense inhibitory oligonucleotides caused marked resistance to h(2)o(2). through bioinformatics, we identified the potential target sites for mir-1 on the 3' utr of bcl-2. mir-1 significantly reduced the expression of bcl-2 in the levels of mrna and protein. the post-transcriptional repression of bcl-2 was further confirmed by luciferase reporter experiments. these data demonstrated that mir-1 plays an important role in the regulation of cardiomyocyte apoptosis, which is involved in post-transcriptional repression of bcl-2.
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endothelial cell migration induced in response to vascular endothelial growth factor (vegf) is a crucial step of angiogenesis and it depends on the activation of the p38 map-kinase pathway downstream of vegfr2. in this study, we investigated the role of micrornas (mirnas) in regulating these processes. we found that the vegf-induced p38 activation and cell migration are modulated by overexpression of argonaute 2, a key protein in the functioning of mirnas. thereafter, we found that mir-20a expression is increased by vegf and that its ectopic expression inhibits vegf-induced actin remodeling and cell migration. moreover, the expression of mir-20a impairs the formation of branched capillaries in a tissue-engineered model of angiogenesis. in addition, the lentivirus-mediated expression of mir-20a precursor (pmir-20a) is associated with a decrease in the vegf-induced activation of p38. in contrast, these processes are increased by inhibiting mir-20a with a specific antagomir. interestingly, mir-20a does not modulate vegfr2 or p38 protein expression level. mir-20a does not affect either the expression of other known actors of the p38 map kinase pathway except mkk3. indeed, by using quantitative pcr and western blot analysis, we found that pmir-20a decreases the expression of mkk3 and we obtained evidence indicating that mir-20a specifically binds to the 3'utr region of mkk3 mrna. in accordance, the vegf-induced activation of p38 and cell migration are impaired when the mkk3 expression is knocked down by sirna. we conclude that mir-20a acts in a feedback loop to repress the expression of mkk3 and to negatively regulate the p38 pathway-mediated vegf-induced endothelial cell migration and angiogenesis.
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gammaherpesviruses, including the human pathogens epstein-barr virus (ebv) and kaposi's sarcoma-associated herpesvirus (kshv), establish lifelong latent infection in b cells and are associated with a variety of tumors. in addition to protein coding genes, these viruses encode numerous micrornas (mirnas) within their genomes. while putative host targets of ebv and kshv mirnas have been previously identified, the specific functions of these mirnas during in vivo infection are largely unknown. murine gammaherpesvirus 68 (mhv68) is a natural pathogen of rodents that is genetically related to both ebv and kshv, and thus serves as an excellent model for the study of ebv and kshv genetic elements such as mirnas in the context of infection and disease. however, the specific targets of mhv68 mirnas remain completely unknown. using a technique known as qclash (quick crosslinking, ligation, and sequencing of hybrids), we have now identified thousands of ago-associated, direct mirna-mrna interactions during lytic infection, latent infection and reactivation from latency. validating this approach, detailed molecular analyses of specific interactions demonstrated repression of numerous host mrna targets of mhv68 mirnas, including arid1a, ctsl, ifitm3 and phc3. notably, of the 1,505 mhv68 mirna-host mrna targets identified in b cells, 86% were shared with either ebv or kshv, and 64% were shared among all three viruses, demonstrating significant conservation of gammaherpesvirus mirna targeting. pathway analysis of mhv68 mirna targets further revealed enrichment of cellular pathways involved in protein synthesis and protein modification, including eif2 signaling, mtor signaling and protein ubiquitination, pathways also enriched for targets of ebv and kshv mirnas. these findings provide substantial new information about specific targets of mhv68 mirnas and shed important light on likely conserved functions of gammaherpesvirus mirnas.
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mirnas silence gene expression by repressing translation and/or by promoting mrna decay. in animal cells, degradation of partially complementary mirna targets occurs via deadenylation by the caf1-ccr4-not1 deadenylase complex, followed by decapping and subsequent exonucleolytic digestion. to determine how generally mirnas trigger deadenylation, we compared mrna expression profiles in d. melanogaster cells depleted of ago1, caf1, or not1. we show that approximately 60% of ago1 targets are regulated by caf1 and/or not1, indicating that deadenylation is a widespread effect of mirna regulation. however, neither a poly(a) tail nor mrna circularization are required for silencing, because mrnas whose 3' ends are generated by a self-cleaving ribozyme are also silenced in vivo. we show further that mirnas trigger mrna degradation, even when binding by 40s ribosomal subunits is inhibited in cis. these results indicate that mirnas promote mrna decay by altering mrnp composition and/or conformation, rather than by directly interfering with the binding and function of ribosomal subunits.
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the overexpression of the hmga1 proteins is a feature of human malignant neoplasias and has a causal role in cell transformation. the aim of our study has been to investigate the micrornas (mirnas or mirs) regulated by the hmga1 proteins in the process of cell transformation analyzing the mirna expression profile of v-ras-ki oncogene-transformed thyroid cells expressing or not hmga1 proteins. we demonstrate that, among the mirnas regulated by cell transformation, there are mir-10b, mir-21, mir-125b, mir-221 and mir-222 that are positively and mir-34a and mir-603 that are negatively regulated by hmga1 expression. then, we focused our attention on the mir-10b and mir-603 whose expression was dependent on the presence of hmga1 also in other cell systems. we found that mir-10b is able to target the pten gene, whereas mir-603 targets the ccnd1 and ccnd2 genes coding for the cyclin d1 and cyclin d2 proteins, respectively. moreover, functional studies showed that mir-10b and mir-603 regulate positively and negatively, respectively, cell proliferation and migration suggesting a role of their dysregulation in thyroid cell transformation.
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hypoxia-induced pulmonary hypertension (ph), which is characterized by vasoconstriction and subsequent structural remodeling of blood vessels, is an important event in chronic obstructive pulmonary disease patients and in people living at high altitudes. hypoxia-inducible factor-1α (hif-1α) and its regulator four-and-a-half lim (lin-11, isl-1 and mec-3) domain 1 (fhl-1) have important roles in hypoxia-induced ph. microrna-206 (mir-206) is critical for myogenesis and related diseases; however, the role of mir-206 in hypoxia-induced ph is unknown. mir-206 expression was evaluated in a hypoxic rat model and in cultured hypoxic pulmonary artery smooth muscle cells (pasmcs) using real-time quantitative pcr (rt-qpcr). hif-1α and fhl-1 expression were evaluated using rt-qpcr, western blotting, immunohistochemistry and immunofluorescence. the function of mir-206 was assessed by transfecting mir-206 mimics and inhibitors. dual-luciferase reporter gene assays and western blotting were performed to validate the target genes of mir-206. sirna targeted against fhl-1 was used to investigate the effect of fhl-1 on mir-206. flow cytometry was used to detect the cell cycle phase distribution in each group of pasmcs. significant downregulation of mir-206 in hypoxic lung tissue and pasmcs was identified, whereas hif-1α and fhl-1 were upregulated in these samples. the expression of mir-206 in the serum was different from that in the lung tissue. transfection of pre-mir mir-206 in hypoxic conditions led to increased expression of hif-1α and fhl-1 rather than abolishing hypoxia-induced hif-1α and fhl-1, as was expected, and promoted the entry of cells into the s phase and enhanced pasmc proliferation. fhl-1-targeted sirna in pasmc prevented cell proliferation and led to an increased proportion of cells in the g1 phase without altering mir-206 expression. bioinformatic analysis and dual-luciferase reporter gene assays revealed direct evidence for mir-206 targeting of hif-1α. in conclusion, hypoxia-induced downregulation of mir-206 promotes ph by targeting the hif-1α/fhl-1 pathway in pasmcs. mir-206 could be a triggering factor of early stage of hypoxia-induced ph.
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respiratory syncytial virus (rsv) causes substantial morbidity and life-threatening lower respiratory tract disease in infants, young children and the elderly. understanding the host response to rsv infection is critical for developing disease-intervention approaches. the role of micrornas (mirnas) in post-transcriptional regulation of host genes responding to rsv infection is not well understood. in this study, it was shown that rsv infection of a human alveolar epithelial cell line (a549) induced five mirnas (let-7f, mir-24, mir-337-3p, mir-26b and mir-520a-5p) and repressed two mirnas (mir-198 and mir-595), and showed that rsv g protein triggered let-7f expression. luciferase-untranslated region reporters and mirna mimics and inhibitors validated the predicted targets, which included cell-cycle genes (ccnd1, dyrk2 and elf4), a chemokine gene (ccl7) and the suppressor of cytokine signalling 3 gene (socs3). modulating let-7 family mirna levels with mirna mimics and inhibitors affected rsv replication, indicating that rsv modulates host mirna expression to affect the outcome of the antiviral host response, and this was mediated in part through rsv g protein expression.
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an internal ribosome entry segment (ires) has been identified in the 5' untranslated region (5' utr) of two members of the myc family of proto-oncogenes, c-myc and n-myc. hence, the synthesis of c-myc and n-myc polypeptides can involve the alternative mechanism of internal initiation. here, we show that the 5' utr of l-myc, another myc family member, also contains an ires. previous studies have shown that the translation of mrnas containing the c-myc and n-myc iress can involve both cap-dependent initiation and internal initiation. in contrast, the data presented here suggest that internal initiation can account for all of the translation initiation that occurs on an mrna with the l-myc ires in its 5' utr. like many other cellular iress, the l-myc ires appears to be modular in nature and the entire 5' utr is required for maximum ires efficiency. the ribosome entry window within the l-myc ires is located some distance upstream of the initiation codon, and thus, this ires uses a "land and scan" mechanism to initiate translation. finally, we have derived a secondary structural model for the ires. the model confirms that the l-myc ires is highly structured and predicts that a pseudoknot may form near the 5' end of the mrna.
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single micrornas are usually associated with hundreds of putative target genes that can influence multiple phenotypic traits in drosophila, ranging from development to behaviour. we investigated the function of drosophila mir-210 in circadian behaviour by misexpressing it within circadian clock cells. manipulation of mir-210 expression levels in the pdf (pigment dispersing factor) positive neurons affected the phase of locomotor activity, under both light-dark conditions and constant darkness. per cyclical expression was not affected in clock neurons, however, when mir-210 was up-regulated, a dramatic alteration in the morphology of pdf ventral lateral neuron (lnv) arborisations was observed. the effect of mir-210 in shaping neuronal projections was confirmed in vitro, using a drosophila neuronal cell line. a transcriptomic analysis revealed that mir-210 overexpression affects the expression of several genes belonging to pathways related to circadian processes, neuronal development, gtpases signal transduction and photoreception. collectively, these data reveal the role of mir-210 in modulating circadian outputs in flies and guiding/remodelling pdf positive lnv arborisations and indicate that mir-210 may have pleiotropic effects on the clock, light perception and neuronal development.
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background pulmonary hypertension (ph) is a proliferative disorder associated with enhanced proliferation and suppressed apoptosis of pulmonary artery smooth muscle cells (pasmcs). our lately study demonstrated that let-7g inhibited hypoxia-induced proliferation of pasmcs via repressing c-myc-bmi-1-p16 signaling pathway. however, the upstream of let-7g has not yet been fully defined. previous studies have shown that lox-1, a target of let-7g, could also regulate the expression of let-7g in human aortic endothelial cells. in this present study, we aimed to investigate whether there is a negative feedback regulation between microrna let-7g and lox-1 in hypoxia-induced proliferation of pasmcs. methods sd rats were exposed to hypoxia (10% o 2 , 3 weeks) to induce ph. he staining was used to evaluate pulmonary artery remodeling. in situ hybridization and immunohistochemistry were performed to assess the expression and distribution of let-7g and lox-1, respectively. mts, edu and flow cytometry were performed to evaluate pasmcs proliferation. quantitative real-time polymerase chain reaction (qrt-pcr) and western blotting were conducted to assess the expression of let-7g, lox-1, calpain-1,-2,-4, and oct-1. results the expression of let-7g was significantly down-regulated in pulmonary arteries of hypoxia-induced ph rats accompanied by pulmonary vascular remodeling, whereas let-7g mimic inhibited hypoxia-induced proliferation of pasmcs and up-regulation of lox-1 expression. lox-1 blocking reversed hypoxia-induced down-regulation of let-7g expression. calpains, protein kinase c and oct-1 were involved in negative feedback regulation between let-7g and lox-1. conclusion negative feedback regulation between let-7g and lox-1 mediated hypoxia-induced proliferation of in pasmcs.
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many molecular pathways involved in heart disease have their roots in evolutionarily ancient developmental programs that depend critically on gene dosage and timing. micrornas (mirnas) modulate gene dosage posttranscriptionally, and among these, the muscle-specific mir-1 is particularly important for developing and maintaining somatic/skeletal and cardiac muscle. to identify pathways regulated by mir-1, we performed a forward genetic screen in drosophila using wing-vein patterning as a biological assay. we identified several unexpected genes that genetically interacted with dmir-1, one of which was kayak, encodes a developmentally regulated transcription factor. additional studies directed at this genetic relationship revealed a previously unappreciated function of dmir-1 in regulating the polarity of cardiac progenitor cells. the mammalian ortholog of kayak, c-fos, was dysregulated in hearts of gain- or loss-of-function mir-1 mutant mice in a stress-dependent manner. these findings illustrate the power of drosophila-based screens to find points of intersection between mirnas and conserved pathways in mammals.
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aims atherosclerosis is the most common cause of cardiovascular disease, such as myocardial infarction and stroke. previous study revealed that microrna (mir)-134 promotes lipid accumulation and proinflammatory cytokine secretion through angiopoietin-like 4 (angptl4)/lipid lipoprotein (lpl) signaling in thp-1 macrophages. methods apoe ko male mice on a c57bl/6 background were fed a high-fat/high-cholesterol western diet, from 8 to 16 weeks of age. mice were divided into four groups, and received a tail vein injection of mir-134 agomir, mir-134 antagomir, or one of the corresponding controls, respectively, once every 2 weeks after starting the western diet. after 8 weeks we measured aortic atherosclerosis, lpl activity, mrna and protein levels of angptl4 and lpl, lpl/ low-density lipoprotein receptor related protein 1 complex formation, proinflammatory cytokine secretion and lipid levels. results despite this finding, the influence of mir-134 on atherosclerosis in vivo remains to be determined. using the well-characterized mouse atherosclerosis model of apolipoprotein e knockout, we found that systemic delivery of mir-134 agomir markedly enhanced the atherosclerotic lesion size, together with a significant increase in proinflammatory cytokine secretion and peritoneal macrophages lipid contents. moreover, overexpression of mir-134 decreased angptl4 expression but increased lpl expression and activity in both aortic tissues and peritoneal macrophages, which was accompanied by increased formation of lpl/low-density lipoprotein receptor-related protein 1 complexes in peritoneal macrophages. however, an opposite effect was observed in response to mir-134 antagomir. conclusions these findings suggest that mir-134 accelerates atherogenesis by promoting lipid accumulation and proinflammatory cytokine secretion via the angptl4/lpl pathway. therefore, targeting mir-134 may offer a promising strategy for the prevention and treatment of atherosclerotic cardiovascular disease.
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the mechanisms by which deregulated nuclear factor erythroid-2-related factor 2 (nrf2) and kelch-like ech-associated protein 1 (keap1) signaling promote cellular proliferation and tumorigenesis are poorly understood. using an integrated genomics and ¹³c-based targeted tracer fate association (ttfa) study, we found that nrf2 regulates mir-1 and mir-206 to direct carbon flux toward the pentose phosphate pathway (ppp) and the tricarboxylic acid (tca) cycle, reprogramming glucose metabolism. sustained activation of nrf2 signaling in cancer cells attenuated mir-1 and mir-206 expression, leading to enhanced expression of ppp genes. conversely, overexpression of mir-1 and mir-206 decreased the expression of metabolic genes and dramatically impaired nadph production, ribose synthesis, and in vivo tumor growth in mice. loss of nrf2 decreased the expression of the redox-sensitive histone deacetylase, hdac4, resulting in increased expression of mir-1 and mir-206, and not only inhibiting ppp expression and activity but functioning as a regulatory feedback loop that repressed hdac4 expression. in primary tumor samples, the expression of mir-1 and mir-206 was inversely correlated with ppp gene expression, and increased expression of nrf2-dependent genes was associated with poor prognosis. our results demonstrate that microrna-dependent (mirna-dependent) regulation of the ppp via nrf2 and hdac4 represents a novel link between mirna regulation, glucose metabolism, and ros homeostasis in cancer cells.
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mir-155 acts as an oncogenic mir in b-cell lymphoproliferative disorders, including waldenstrom macroglobulinemia (wm) and chronic lymphocytic leukemia, and is therefore a potential target for therapeutic intervention. however, efficient targeting of mirs in tumor cells in vivo remains a significant challenge for the development of mir-155-based therapeutics for the treatment of b-cell malignancies. in the present study, we show that an 8-mer locked nucleic acid anti-mir-155 oligonucleotide targeting the seed region of mir-155 inhibits wm and chronic lymphocytic leukemia cell proliferation in vitro. moreover, anti-mir-155 delivered systemically showed uptake in the bm cd19(+) cells of wm-engrafted mice, resulting in the up-regulation of several mir-155 target mrnas in these cells, and decreased tumor growth significantly in vivo. we also found mir-155 levels to be elevated in stromal cells from wm patients compared with control samples. interestingly, stromal cells from mir-155-knockout mice led to significant inhibition of wm tumor growth, indicating that mir-155 may also contribute to wm proliferation through bm microenvironmental cells. the results of the present study highlight the therapeutic potential of anti-mir-155-mediated inhibition of mir-155 in the treatment of wm.
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chronic lymphocytic leukemia (cll) is the most common human leukemia and is characterized by predominantly nondividing malignant b cells overexpressing the antiapoptotic b cell lymphoma 2 (bcl2) protein. mir-15a and mir-16-1 are deleted or down-regulated in the majority of clls. here, we demonstrate that mir-15a and mir-16-1 expression is inversely correlated to bcl2 expression in cll and that both micrornas negatively regulate bcl2 at a posttranscriptional level. bcl2 repression by these micrornas induces apoptopsis in a leukemic cell line model. therefore, mir-15 and mir-16 are natural antisense bcl2 interactors that could be used for therapy of bcl2-overexpressing tumors.
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the nonselective β-adrenergic receptor antagonist (β-blocker) carvedilol has been shown to protect against myocardial injury, but the detailed underlying mechanisms are unclear. we recently reported that carvedilol stimulates the processing of microrna (mir)-199a-3p and mir-214 in the heart via β-arrestin1-biased β1-adrenergic receptor (β1ar) cardioprotective signaling. here, we investigate whether these β-arrestin1/β1ar-responsive mirs mediate the beneficial effects of carvedilol against simulated ischemia/reperfusion (si/r). using cultured cardiomyocyte cell lines and primary cardiomyocytes, we demonstrate that carvedilol upregulates mir-199a-3p and mir-214 in both ventricular and atrial cardiomyocytes subjected to si/r. overexpression of the two mirs in cardiomyocytes mimics the effects of carvedilol to activate p-akt survival signaling and the expression of a downstream pluripotency marker sox2 in response to si/r. moreover, carvedilol-mediated p-akt activation is abolished by knockdown of either mir-199a-3p or mir-214. along with previous studies to directly link the cardioprotective actions of carvedilol to upregulation of p-akt/stem cell markers, our findings suggest that the protective roles of carvedilol during ischemic injury are in part attributed to activation of these two protective mirs. loss of function of mir-199a-3p and mir-214 also increases cardiomyocyte apoptosis after si/r. mechanistically, we demonstrate that mir-199a-3p and mir-214 repress the predictive or known apoptotic target genes ddit4 and ing4, respectively, in cardiomyocytes. these findings suggest pivotal roles for mir-199a-3p and mir-214 as regulators of cardiomyocyte survival and contributors to the functional benefits of carvedilol therapy.
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nuclear factor of activated t cells (nfat) proteins are ca(2+)-regulated transcription factors that control gene expression in many cell types. nfat proteins are heavily phosphorylated and reside in the cytoplasm of resting cells; when cells are stimulated by a rise in intracellular ca(2+), nfat proteins are dephosphorylated by the ca(2+)/calmodulin-dependent phosphatase calcineurin and translocate to the nucleus to activate target gene expression. here we show that phosphorylated nfat1 is present in a large cytoplasmic rna-protein scaffold complex that contains a long intergenic noncoding rna (lincrna), nron ; a scaffold protein, iq motif containing gtpase activating protein (iqgap); and three nfat kinases, casein kinase 1, glycogen synthase kinase 3, and dual specificity tyrosine phosphorylation regulated kinase. combined knockdown of nron and iqgap1 increased nfat dephosphorylation and nuclear import exclusively after stimulation, without affecting the rate of nfat rephosphorylation and nuclear export; and both nron-depleted t cells and t cells from iqgap1-deficient mice showed increased production of nfat-dependent cytokines. our results provide evidence that a complex of lincrna and protein forms a scaffold for a latent transcription factor and its regulatory kinases, and support an emerging consensus that lincrnas that bind transcriptional regulators have a similar scaffold function.
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micrornas (mirnas) are short noncoding rnas, which post-transcriptionally regulate gene expression. mirnas are transcribed as precursors and matured to active forms by a series of enzymes, including dicer. mirnas are important in governing cell differentiation, development, and disease. we have recently developed a feeder- and serum-free protocol for direct derivation of endothelial cells (ecs) from human embryonic stem cells (hescs) and provided evidence of increases in angiogenesis-associated mirnas (mir-126 and -210) during the process. however, the functional role of mirnas in hesc differentiation to vascular ec remains to be fully interrogated. here, we show that the reduction of mirna maturation induced by dicer knockdown suppressed hes-ec differentiation. a mirna microarray was performed to quantify hes-ec mirna profiles during defined stages of endothelial differentiation. mir-99b, -181a, and -181b were identified as increasing in a time- and differentiation-dependent manner to peak in mature hesc-ecs and adult ecs. augmentation of mir-99b, -181a, and -181b levels by lentiviral-mediated transfer potentiated the mrna and protein expression of ec-specific markers, pecam1 and ve cadherin, increased nitric oxide production, and improved hes-ec-induced therapeutic neovascularization in vivo. conversely, knockdown did not impact endothelial differentiation. our results suggest that mir-99b, -181a, and -181b comprise a component of an endothelial-mirna signature and are capable of potentiating ec differentiation from pluripotent hescs.
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the transcriptional repressor bcl6 is a critical regulator of t helper cell fate, and inhibits th2-type inflammation. we have found that microrna-21 (mir-21) is a novel target gene for bcl6 in treg cells. bcl6 represses and stat3 activates mir-21 transcription through a stat3 binding element in the promoter, indicating opposing regulation of mir-21 by the two transcription factors via the same dna site. ectopic expression of mir-21 promoted th2 differentiation in non-polarized t cells. the pro-th2 activity of mir-21 was associated with increased gata3 expression and decreased expression of the mir-21 target gene sprouty1. increased mir-21 promoted th2 and treg gene expression in wild-type tregs. mir-21 could thus help promote the th2 bias of bcl6-deficient conventional t cells and treg cells. mir21 expression is increased in th2-type inflammation, and our results define mir-21 as a critical target of bcl6, thus providing a new link between bcl6 and th2 inflammation. finally, our results reveal a novel t cell autonomous role for mir-21 in promoting th2 differentiation.
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the transcriptional networks that regulate embryonic stem (es) cell pluripotency and lineage specification are the subject of considerable attention. to date such studies have focused almost exclusively on protein-coding transcripts. however, recent transcriptome analyses show that the mammalian genome contains thousands of long noncoding rnas (ncrnas), many of which appear to be expressed in a developmentally regulated manner. the functions of these remain untested. to identify ncrnas involved in es cell biology, we used a custom-designed microarray to examine the expression profiles of mouse es cells differentiating as embryoid bodies (ebs) over a 16-d time course. we identified 945 ncrnas expressed during eb differentiation, of which 174 were differentially expressed, many correlating with pluripotency or specific differentiation events. candidate ncrnas were identified for further characterization by an integrated examination of expression profiles, genomic context, chromatin state, and promoter analysis. many ncrnas showed coordinated expression with genomically associated developmental genes, such as dlx1, dlx4, gata6, and ecsit. we examined two novel developmentally regulated ncrnas, evx1as and hoxb5/6as, which are derived from homeotic loci and share similar expression patterns and localization in mouse embryos with their associated protein-coding genes. using chromatin immunoprecipitation, we provide evidence that both ncrnas are associated with trimethylated h3k4 histones and histone methyltransferase mll1, suggesting a role in epigenetic regulation of homeotic loci during es cell differentiation. taken together, our data indicate that long ncrnas are likely to be important in processes directing pluripotency and alternative differentiation programs, in some cases through engagement of the epigenetic machinery.
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cooperation between dna, rna and protein regulates gene expression and controls differentiation through interactions that connect regions of nucleic acids and protein domains and through the assembly of biomolecular condensates. here, we report that endoderm differentiation is regulated by the interaction between the long non-coding rna (lncrna) digit and the bromodomain and extraterminal domain protein brd3. brd3 forms phase-separated condensates of which the formation is promoted by digit, occupies enhancers of endoderm transcription factors and is required for endoderm differentiation. brd3 binds to histone h3 acetylated at lysine 18 (h3k18ac) in vitro and co-occupies the genome with h3k18ac. digit is also enriched in regions of h3k18ac, and the depletion of digit results in decreased recruitment of brd3 to these regions. our findings show that cooperation between digit and brd3 at regions of h3k18ac regulates the transcription factors that drive endoderm differentiation and suggest that protein-lncrna phase-separated condensates have a broader role as regulators of transcription.
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obesity, type 2 diabetes, and heart failure are associated with aberrant cardiac metabolism. we show that the heart regulates systemic energy homeostasis via med13, a subunit of the mediator complex, which controls transcription by thyroid hormone and other nuclear hormone receptors. med13, in turn, is negatively regulated by a heart-specific microrna, mir-208a. cardiac-specific overexpression of med13 or pharmacologic inhibition of mir-208a in mice confers resistance to high-fat diet-induced obesity and improves systemic insulin sensitivity and glucose tolerance. conversely, genetic deletion of med13 specifically in cardiomyocytes enhances obesity in response to high-fat diet and exacerbates metabolic syndrome. the metabolic actions of med13 result from increased energy expenditure and regulation of numerous genes involved in energy balance in the heart. these findings reveal a role of the heart in systemic metabolic control and point to med13 and mir-208a as potential therapeutic targets for metabolic disorders.
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in caenorhabditis elegans, the heterochronic pathway controls the timing of developmental events during the larval stages. a component of this pathway, the let-7 small regulatory rna, is expressed at the late stages of development and promotes the transition from larval to adult (l/a) stages. the stage-specificity of let-7 expression, which is crucial for the proper timing of the worm l/a transition, is conserved in drosophila melanogaster and other invertebrates. in drosophila, pulses of the steroid hormone 20-hydroxyecdysone (ecdysone) control the timing of the transition from larval to pupal to adult stages. to test whether let-7 expression is regulated by ecdysone in drosophila, we used northern blot analysis to examine the effect of altered ecdysone levels on let-7 expression in mutant animals, organ cultures, and s2 cultured cells. experiments were conducted to test the role of broad-complex (br-c), an essential component in the ecdysone pathway, in let-7 expression. we show that ecdysone and br-c are required for let-7 expression, indicating that the ecdysone pathway regulates the temporal expression of let-7 in drosophila. these results demonstrate an interaction between steroid hormone signaling and the heterochronic pathway in insects.
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both micrornas and alternative pre-mrna splicing have been implicated in the development of the nervous system (ns), but functional interactions between these two pathways are poorly understood. we demonstrate that the neuron-specific microrna mir-124 directly targets ptbp1 (ptb/hnrnp i) mrna, which encodes a global repressor of alternative pre-mrna splicing in nonneuronal cells. among the targets of ptbp1 is a critical cassette exon in the pre-mrna of ptbp2 (nptb/brptb/ptblp), an ns-enriched ptbp1 homolog. when this exon is skipped, ptbp2 mrna is subject to nonsense-mediated decay (nmd). during neuronal differentiation, mir-124 reduces ptbp1 levels, leading to the accumulation of correctly spliced ptbp2 mrna and a dramatic increase in ptbp2 protein. these events culminate in the transition from non-ns to ns-specific alternative splicing patterns. we also present evidence that mir-124 plays a key role in the differentiation of progenitor cells to mature neurons. thus, mir-124 promotes ns development, at least in part by regulating an intricate network of ns-specific alternative splicing.
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in prokaryotes, small noncoding rnas (snrnas) of 50-500 nt are produced that are important in bacterial virulence and response to environmental stimuli. here, we identified and characterized snrnas from the endosymbiotic bacteria, wolbachia, which are widespread in invertebrates and cause reproductive manipulations. most importantly, some strains of wolbachia inhibit replication of several vector-borne pathogens in insects. we demonstrate that two abundant snrnas, wsnrna-46 and wsnrna-49, are expressed in wolbachia from noncoding rna transcripts that contain precursors with stem-loop structures. wsnrnas were detected in aedes aegypti mosquitoes infected with the wmelpop-cla strain of wolbachia and in drosophila melanogaster and drosophila simulans infected with wmelpop and wau strains, respectively, indicating that the wsnrnas are conserved across species and strains. in addition, we show that the wsnrnas may potentially regulate host genes and wolbachia genes. our findings provide evidence for the production of functional snrnas by wolbachia that play roles in cross-kingdom communication between the endosymbiont and the host.
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hedgehog (hh) signaling plays many important roles in developmental processes and cancers. smoothened (smo) is an important signal transducer in the hh pathway, and its expression is tightly regulated by several different post-transcriptional mechanisms. however, whether micrornas (mirnas) are involved in smo regulation is still unclear. here, we found that mir-5 acts as a suppressor of the hh pathway by targeting smo. through in vivo sensor assay and in vitro luciferase assay, we found that mir-5 downregulates smo through directly binding to its 3'utr. moreover, our data indicated costal-2 (cos2) and fused (fu) do not play a role in the reduction of smo mediated by mir-5. furthermore, we determined that mir-5 not involved in notch or dpp signaling pathways by detecting target gene expression. together, our results indicate that mir-5 can specifically suppress hh signaling by directly targeting smo in drosophila.
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recently, researchers have uncovered the presence of many long noncoding rnas (lncrnas) in embryonic stem cells and believe they are important regulators of the differentiation process. however, there are only a few examples explicitly linking lncrna activity to transcriptional regulation. here, we used transcript counting and spatial localization to characterize a lncrna (dubbed linc-hoxa1) located ∼50 kb from the hoxa gene cluster in mouse embryonic stem cells. single-cell transcript counting revealed that linc-hoxa1 and hoxa1 rna are highly variable at the single-cell level and that whenever linc-hoxa1 rna abundance was high, hoxa1 mrna abundance was low and vice versa. knockdown analysis revealed that depletion of linc-hoxa1 rna at its site of transcription increased transcription of the hoxa1 gene cis to the chromosome and that exposure of cells to retinoic acid can disrupt this interaction. we further showed that linc-hoxa1 rna represses hoxa1 by recruiting the protein purb as a transcriptional cofactor. our results highlight the power of transcript visualization to characterize lncrna function and also suggest that purb can facilitate lncrna-mediated transcriptional regulation.
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once protein-coding, the x-inactivation center (xic) is now dominated by large noncoding rnas (ncrna). x chromosome inactivation (xci) equalizes gene expression between mammalian males and females by inactivating one x in female cells. xci requires xist, an ncrna that coats the x and recruits polycomb proteins. how xist is controlled remains unclear but likely involves negative and positive regulators. for the active x, the antisense tsix rna is an established xist repressor. for the inactive x, here, we identify xic-encoded jpx as an xist activator. jpx is developmentally regulated and accumulates during xci. deleting jpx blocks xci and is female lethal. posttranscriptional jpx knockdown recapitulates the knockout, and supplying jpx in trans rescues lethality. thus, jpx is trans-acting and functions as ncrna. furthermore, δjpx is rescued by truncating tsix, indicating an antagonistic relationship between the ncrnas. we conclude that xist is controlled by two rna-based switches: tsix for xa and jpx for xi.
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while recent insights indicate that the transcription factor krüppel-like factor 4 (klf4) is indispensable for vascular homeostasis, its exact role in proliferation and angiogenesis and how it functions remain unresolved. thus, the aim of the present study was to evaluate the role of klf4 in the proliferations of endothelial and vascular smooth muscle cells, as well as the angiogenesis. the overexpression of klf4 in endothelial cells significantly impaired tube formation. klf4 inhibited the formation of a vascular network in implanted matrigel plugs in nude mice. importantly, we found that klf4 significantly upregulated the mir-15a expression in endothelial cells and vascular smooth muscle cells, and conversely, klf4 depletion reduced the amount of mir-15a. furthermore, klf4 blocked cell cycle progression and decreased cyclin d1 expression in endothelial cells and vascular smooth muscle cells through the induction of mir-15a. intriguingly, the delivery of a mir-15a antagomir to nude mice resulted in marked attenuation of the anti-angiogenic effect of klf4. collectively, our present study provide the first evidence that mir-15a as a direct transcriptional target of klf4 that mediates the anti-proliferative and anti-angiogenic actions of klf4, which indicates that klf4 upregulation of mir-15a may represent a therapeutic option to suppress proliferative vascular disorders.
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RNAcentral Litscan ncRNA Related Abstracts
This is a dataset of abstracts that have been labelled relevant to ncRNA or not. Relevant articles have the label 1, irrelevant have the label 0
How was it made
The dataset was built in five parts:
- ncRNA related articles from tarbase
- ncRNA related articles from Rfam
- ncRNA related articles from GO curation
- Manually annotated ncRNA related articles
- Non ncRNA related articles
TarBase
TarBase is a database of miRNAs and their targets. This data is curated from the literature, so by definition, the papers they cite for the interactions are relevant for ncRNA. This is somewhat limited to miRNA though
Rfam
Rfam is the ncRNA families database, and has families for >4000 RNAs. They are mostly miRNAs, but also cover a lot of other types, and the non-miRNA part is expanding. Rfam families are usually built from an alignmentthat is published, so the papers mentioned by Rfam families will be relevant to ncRNA
GO
GO is the Gene Ontology, a knowledge base of function in biology. A subset of annotations in GO are manually curated by human curators reading papers, so those papers curated with ncRNA relevant terms will definitely be relevant to ncRNA. A large fraction of the GO curation is about miRNA-mRNA interactions, but there is a bit of other stuff in there as well; reflecting the type of analyses done in low throughput experiments on ncRNA
Manually annotated articles
A set of ~400 abstracts were manually assessed for relevance to ncRNA by members of the RNAcentral & Rfam teams. These were drawn from a EuropePMC query designed to pull in as many potentially relevant articles as possible. These should cover all RNA types, but the set is relatively small
Non-ncRNA related articles
To provide our negative set, we used this EuropePMC search to retrieve ~3500 articles:
f'/search?query=(IN_EPMC:Y AND OPEN_ACCESS:Y AND NOT SRC:PPR AND NOT "rna" ' \
f'AND NOT "mrna" AND NOT "ncrna" AND NOT "lncrna" AND NOT "rrna" AND NOT "sncrna" AND NOT "mirna") ' \
f'&sort_cited:y&pageSize=500&cursorMark={page}&format=json'
This is the opposite query to the one used in production for LitScan, so should be pulling in totally irrelevant articles.
Limitations
- Most of the positive set come from annotations or other linkages with miRNAs. Therefore, we kind of expect this dataset to produce a model that is good at finding miRNA related articles, but it may struggle when filtering articles relevant for other types (e.g. snoRNA) that are under-represented
- The negative set may be too easy to separate from the positive, since they are deliberately very irrelevant
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