An APOA5 3′ UTR Variant Associated with Plasma Triglycerides Triggers APOA5 Downregulation by Creating a Functional miR-485-5p Binding Site
2014; Elsevier BV; Volume: 94; Issue: 1 Linguagem: Inglês
10.1016/j.ajhg.2013.12.001
ISSN1537-6605
AutoresCyrielle Caussy, Sybil Charrière, Christophe Marçais, Mathilde Di Filippo, Agnès Sassolas, Mireille Delay, Vanessa Euthine, Audrey Jalabert, Étienne Lefai, Sophie Rome, Philippe Moulin,
Tópico(s)RNA modifications and cancer
ResumoAPOA5 c.∗158C>T (rs2266788), located in the 3′ UTR, belongs to APOA5 haplotype 2 (APOA5∗2), which is strongly associated with plasma triglyceride levels and modulates the occurrence of both moderate and severe hypertriglyceridemia. Individuals with APOA5∗2 display reduced APOA5 expression at the posttranscriptional level. However, the functionality of this haplotype remains unclear. We hypothesized that the hypertriglyceridemic effects of APOA5∗2 could involve miRNA regulation in the APOA5 3′ UTR. Bioinformatic studies have identified the creation of a potential miRNA binding site for liver-expressed miR-485-5p (MIRN485-5p) in the mutant APOA5 3′ UTR with the c.∗158C allele. In human embryonic kidney 293T (HEK293T) cells cotransfected with an APOA5 3′ UTR luciferase reporter vector and a miR485-5p precursor, c.∗158C allele expression was significantly decreased. Moreover, in HuH-7 cells endogenously expressing miR-485-5p, we observed that luciferase activity was significantly lower in the presence of the c.∗158C allele than in the presence of the c.∗158T allele, which was completely reversed by a miR-485-5p inhibitor. We demonstrated that the rare c.∗158C APOA5 allele creates a functional target site for liver-expressed miR-485-5p. Therefore, we propose that the well-documented hypertriglyceridemic effect of APOA5∗2 involves an APOA5 posttranscriptional downregulation mediated by miR-485-5p. APOA5 c.∗158C>T (rs2266788), located in the 3′ UTR, belongs to APOA5 haplotype 2 (APOA5∗2), which is strongly associated with plasma triglyceride levels and modulates the occurrence of both moderate and severe hypertriglyceridemia. Individuals with APOA5∗2 display reduced APOA5 expression at the posttranscriptional level. However, the functionality of this haplotype remains unclear. We hypothesized that the hypertriglyceridemic effects of APOA5∗2 could involve miRNA regulation in the APOA5 3′ UTR. Bioinformatic studies have identified the creation of a potential miRNA binding site for liver-expressed miR-485-5p (MIRN485-5p) in the mutant APOA5 3′ UTR with the c.∗158C allele. In human embryonic kidney 293T (HEK293T) cells cotransfected with an APOA5 3′ UTR luciferase reporter vector and a miR485-5p precursor, c.∗158C allele expression was significantly decreased. Moreover, in HuH-7 cells endogenously expressing miR-485-5p, we observed that luciferase activity was significantly lower in the presence of the c.∗158C allele than in the presence of the c.∗158T allele, which was completely reversed by a miR-485-5p inhibitor. We demonstrated that the rare c.∗158C APOA5 allele creates a functional target site for liver-expressed miR-485-5p. Therefore, we propose that the well-documented hypertriglyceridemic effect of APOA5∗2 involves an APOA5 posttranscriptional downregulation mediated by miR-485-5p. Hypertriglyceridemia (HTG [MIM 145750 and 144600]) is a common metabolic disease resulting from complex interactions between genetic and environmental factors.1Marçais C. Bernard S. Merlin M. Ulhmann M. Mestre B. Rochet-Mingret L. Revol A. Berthezene F. Moulin P. Severe hypertriglyceridaemia in Type II diabetes: involvement of apoC-III Sst-I polymorphism, LPL mutations and apo E3 deficiency.Diabetologia. 2000; 43: 1346-1352Crossref PubMed Scopus (29) Google Scholar, 2Johansen C.T. Kathiresan S. Hegele R.A. Genetic determinants of plasma triglycerides.J. 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Billon S. Delay M. et al.Modulation of phenotypic expression of APOA5 Q97X and L242P mutations.Atherosclerosis. 2009; 207: 150-156Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 11Pennacchio L.A. Olivier M. Hubacek J.A. Krauss R.M. Rubin E.M. Cohen J.C. Two independent apolipoprotein A5 haplotypes influence human plasma triglyceride levels.Hum. Mol. Genet. 2002; 11: 3031-3038Crossref PubMed Google Scholar, 12Kraja A.T. Vaidya D. Pankow J.S. Goodarzi M.O. Assimes T.L. Kullo I.J. Sovio U. Mathias R.A. Sun Y.V. Franceschini N. et al.A bivariate genome-wide approach to metabolic syndrome: STAMPEED consortium.Diabetes. 2011; 60: 1329-1339Crossref PubMed Scopus (195) Google Scholar, 13Tan A. Sun J. Xia N. Qin X. Hu Y. Zhang S. Tao S. Gao Y. Yang X. Zhang H. et al.A genome-wide association and gene-environment interaction study for serum triglycerides levels in a healthy Chinese male population.Hum. Mol. 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Determination of the functionality of common APOA5 polymorphisms.J. Biol. Chem. 2005; 280: 28215-28220Crossref PubMed Scopus (107) Google Scholar A second APOA5 haplotype (APOA5∗2) includes the C rare allele of the c.∗158C>T SNP (rs2266788; RefSeq NM_052968.4), which is located in the APOA5 3′ UTR (previously described as APOA5 SNP1, c.1891T>C, or c.1259T>C) and is in strong linkage disequilibrium with three additional SNPs: g.4430C>T (rs662799; RefSeq NG_015894.1; previously described as APOA5 SNP3 or g.−1131T>C), c.−3A>G (rs651821; RefSeq NM_052968.4), and c.162−43A>G (rs2072560; RefSeq NM_052968.4; previously described as APOA5 SNP2).3Pennacchio L.A. Olivier M. Hubacek J.A. Cohen J.C. Cox D.R. Fruchart J.C. Krauss R.M. Rubin E.M. An apolipoprotein influencing triglycerides in humans and mice revealed by comparative sequencing.Science. 2001; 294: 169-173Crossref PubMed Scopus (799) Google Scholar, 11Pennacchio L.A. Olivier M. Hubacek J.A. Krauss R.M. Rubin E.M. Cohen J.C. Two independent apolipoprotein A5 haplotypes influence human plasma triglyceride levels.Hum. Mol. Genet. 2002; 11: 3031-3038Crossref PubMed Google Scholar The frequency of APOA5∗2 is approximately 7% in populations of European descent and is strongly associated with both mild and severe HTG.10Charrière S. Cugnet C. Guitard M. Bernard S. Groisne L. Charcosset M. Pruneta-Deloche V. Merlin M. Billon S. Delay M. et al.Modulation of phenotypic expression of APOA5 Q97X and L242P mutations.Atherosclerosis. 2009; 207: 150-156Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 14Wang J. Ban M.R. Kennedy B.A. Anand S. Yusuf S. Huff M.W. Pollex R.L. Hegele R.A. APOA5 genetic variants are markers for classic hyperlipoproteinemia phenotypes and hypertriglyceridemia.Nat. Clin. Pract. Cardiovasc. Med. 2008; 5: 730-737Crossref PubMed Scopus (49) Google Scholar, 15Charriere S. Bernard S. Aqallal M. Merlin M. Billon S. Perrot L. Le Coquil E. Sassolas A. Moulin P. Marcais C. Association of APOA5 -1131T>C and S19W gene polymorphisms with both mild hypertriglyceridemia and hyperchylomicronemia in type 2 diabetic patients.Clin. Chim. Acta. 2008; 394: 99-103Crossref PubMed Scopus (26) Google Scholar This strong association with plasma TG concentrations was confirmed in genome-wide association studies (GWASs).12Kraja A.T. Vaidya D. Pankow J.S. Goodarzi M.O. Assimes T.L. Kullo I.J. Sovio U. Mathias R.A. Sun Y.V. Franceschini N. et al.A bivariate genome-wide approach to metabolic syndrome: STAMPEED consortium.Diabetes. 2011; 60: 1329-1339Crossref PubMed Scopus (195) Google Scholar, 13Tan A. Sun J. Xia N. Qin X. Hu Y. Zhang S. Tao S. Gao Y. Yang X. Zhang H. et al.A genome-wide association and gene-environment interaction study for serum triglycerides levels in a healthy Chinese male population.Hum. Mol. Genet. 2012; 21: 1658-1664Crossref PubMed Scopus (56) Google Scholar, 17Comuzzie A.G. Cole S.A. Laston S.L. Voruganti V.S. Haack K. Gibbs R.A. Butte N.F. 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Krauss R.M. Rubin E.M. An apolipoprotein influencing triglycerides in humans and mice revealed by comparative sequencing.Science. 2001; 294: 169-173Crossref PubMed Scopus (799) Google Scholar Some studies have also shown a decreased plasma apoAV concentration in subjects with APOA5∗2.20Hahne P. Krempler F. Schaap F.G. Soyal S.M. Höffinger H. Miller K. Oberkofler H. Strobl W. Patsch W. Determinants of plasma apolipoprotein A-V and APOA5 gene transcripts in humans.J. Intern. Med. 2008; 264: 452-462Crossref PubMed Scopus (26) Google Scholar, 21Ishihara M. Kujiraoka T. Iwasaki T. Nagano M. Takano M. Ishii J. Tsuji M. Ide H. Miller I.P. Miller N.E. Hattori H. A sandwich enzyme-linked immunosorbent assay for human plasma apolipoprotein A-V concentration.J. Lipid Res. 2005; 46: 2015-2022Crossref PubMed Scopus (90) Google Scholar, 22Dallinga-Thie G.M. van Tol A. Hattori H. van Vark-van der Zee L.C. Jansen H. Sijbrands E.J. 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Similar APOA5 mRNA levels were found in mice overexpressing human APOA5, either wild-type or with APOA5∗2.25Ahituv N. Akiyama J. Chapman-Helleboid A. Fruchart J. Pennacchio L.A. In vivo characterization of human APOA5 haplotypes.Genomics. 2007; 90: 674-679Crossref PubMed Scopus (23) Google Scholar Moreover, APOA5 mRNA levels were similar in human hepatic biopsies in subjects with either wild-type APOA5 or APOA5∗2.20Hahne P. Krempler F. Schaap F.G. Soyal S.M. Höffinger H. Miller K. Oberkofler H. Strobl W. Patsch W. Determinants of plasma apolipoprotein A-V and APOA5 gene transcripts in humans.J. Intern. Med. 2008; 264: 452-462Crossref PubMed Scopus (26) Google Scholar However, the underlying mechanisms involved in APOA5∗2 dysfunction remain unknown, e.g., Palmen et al. did not confirm in vitro the hypothesis that within APOA5∗2, the c.−3A>G Kozak sequence polymorphism could alter APOA5 mRNA translation.24Palmen J. Smith A.J. Dorfmeister B. Putt W. Humphries S.E. Talmud P.J. The functional interaction on in vitro gene expression of APOA5 SNPs, defining haplotype APOA52, and their paradoxical association with plasma triglyceride but not plasma apoAV levels.Biochim. Biophys. Acta. 2008; 1782: 447-452Crossref PubMed Scopus (36) Google Scholar Consequently, we investigated the potential implication of miRNAs in the posttranscriptional regulation of mutant APOA5. miRNAs are evolutionally conserved 19–22 nucleotides of noncoding RNA that posttranscriptionally downregulate gene expression by binding target mRNAs. This process leads to mRNA degradation or translation repression. Recent data have suggested that miRNAs predominantly decrease mRNA stability through base pairing with the 3′ UTR of target mRNAs. The recognition of target mRNA by miRNA involves a small complementary sequence from 2 to 7 nucleotides long.26Fabian M.R. Sonenberg N. The mechanics of miRNA-mediated gene silencing: a look under the hood of miRISC.Nat. Struct. Mol. 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The PLIN4 variant rs8887 modulates obesity related phenotypes in humans through creation of a novel miR-522 seed site.PLoS ONE. 2011; 6: e17944Crossref PubMed Scopus (48) Google Scholar Within APOA5∗2, c.∗158C>T is the only SNP located in the 3′ UTR. We hypothesized that the APOA5 3′ UTR c.∗158C>T rare variant might create an illegitimate binding site for miRNAs and thus result in APOA5 posttranscriptional inhibition. This could lead to downregulation of lipolysis and a subsequent increase in plasma TG levels. Using five distinct software tools for miRNA target prediction, we performed bioinformatic studies to determine whether c.∗158C>T affects the binding of specific miRNAs (Table S1, available online). First, we used PITA30Kertesz M. Iovino N. Unnerstall U. Gaul U. Segal E. The role of site accessibility in microRNA target recognition.Nat. Genet. 2007; 39: 1278-1284Crossref PubMed Scopus (1894) Google Scholar and RegRNA31Huang H.Y. Chien C.H. Jen K.H. Huang H.D. RegRNA: an integrated web server for identifying regulatory RNA motifs and elements.Nucleic Acids Res. 2006; 34: W429-W434Crossref PubMed Scopus (195) Google Scholar to investigate the potential effect of the c.∗158C allele on miRNA target sites and how it compares to that of the c.∗158T allele. The miRNA-mRNA binding scores were stronger in the presence of the C allele than in the presence of the T allele for three miRNAs: miR-485-5p (MIRN485-5p) and miR-1255a (MIRN1255a) were predicted by the two programs, whereas miR-3188 (MIRN3188) was only predicted by RegRNA (Table S2). Second, we utilized three additional programs (microRNA.org,32Betel D. Wilson M. Gabow A. Marks D.S. Sander C. The microRNA.org resource: targets and expression.Nucleic Acids Res. 2008; 36: D149-D153PubMed Google Scholar TargetScan,33Grimson A. Farh K.K. Johnston W.K. Garrett-Engele P. Lim L.P. Bartel D.P. MicroRNA targeting specificity in mammals: determinants beyond seed pairing.Mol. Cell. 2007; 27: 91-105Abstract Full Text Full Text PDF PubMed Scopus (3031) Google Scholar and Diana-microT34Maragkakis M. Vergoulis T. Alexiou P. Reczko M. Plomaritou K. Gousis M. Kourtis K. Koziris N. Dalamagas T. Hatzigeorgiou A.G. DIANA-microT Web server upgrade supports Fly and Worm miRNA target prediction and bibliographic miRNA to disease association.Nucleic Acids Res. 2011; 39: W145-W148Crossref PubMed Scopus (109) Google Scholar) to confirm the potential binding of these three miRNAs on the c.∗158C allele: miR485-5p binding was confirmed by the three programs, miR3188 binding was confirmed only by microRNA.org, and miR1255a binding was not confirmed (Table S2). Consequently, these results indicate that the c.∗158C allele might create a potential illegitimate target site at least for miR-485-5p. Given the discrepancies between the programs, we decided to also consider miR-1255a and miR-3188 for in vitro binding validation. Luciferase expression vectors containing the C or T allele (pEZX-C or pEZX-T, respectively) were constructed for functional assessment of the binding of the three candidate miRNAs on the mutant APOA5 3′ UTR (Figure 1A). Human embryonic kidney 293T (HEK293T) cells (ATCC CRL-11268) were cotransfected with pEZX-C or pEZX-T and with either a miRNA precursor (miR-485-5p, miR-1255a, or miR-3188) or a control mimic (miR-CTRL). Compared to control miRNA, the APOA5 3′ UTR containing the c.∗158C allele showed a significant, 35% (±6%) decrease in luciferase activity in the presence of miR-485-5p (p < 0.001). Meanwhile, the 3′ UTR containing the c.∗158T allele showed a nonsignificant increase in the level of luciferase activity in the presence of miR-485-5p in comparison to control miRNA (Figure 1A). However, when the c.∗158T or c.∗158C allele was expressed in the presence of miR-1255a (Figure S1A) or miR-3188 (Figure S1B), there was no significant difference in comparison to expression with the miR-CTRL. The specific effect of miR-485-5p on the luciferase activity of the APOA5 3′ UTR c.∗158C allele was assessed with a mir-485-5p inhibitor (anti-485-5p) or a negative control inhibitor (anti-neg). The anti-485-5p fully reversed the decrease in luciferase activity of the APOA5 3′ UTR c.∗158C allele, whereas in the same conditions, the anti-neg had no significant effect (Figure 1B). These results confirm that in vitro miR-485-5p is capable of targeting the APOA5 3′ UTR c.∗158C sequence and of partially repressing its luciferase activity. Because APOA5 is exclusively expressed in the liver, only liver-expressed miRNAs would potentially regulate APOA5 expression. We sought to determine whether miR-485-5p was expressed in the liver by using quantitative RT-PCR on RNA extracted from human hepatic cells (HepG2 [ATCC HB-8065] and HuH-7 [human hepatoma cell line, JCRB-0403]) and mouse and human liver tissue (generous gift from the pathology laboratory of Centre Hospitalier Lyon Sud). miR-485-5p was expressed in both human and mouse hepatic tissue and in all hepatic cell lines tested and was most highly expressed in human hepatic tissue (Figure S2). To confirm our results obtained with HEK293T cells, we investigated endogenous miR-485-5p functionality in human hepatic cell line HuH-7, expressing miR-485-5p. HuH-7 cells were transfected with the luciferase expression vectors pEZX-C or pEZX-T. We confirmed the specific effect of endogenous miR-485-5p by using anti-485-5p or anti-neg cotransfected with pEZX-C or pEZX-T. We observed that the APOA5 3′ UTR c.∗158C allele showed significantly less luciferase activity than did the c.∗158T allele (−43 ± 16%, p < 0.01) (Figure 2A). This decrease was completely reversed in the presence of anti-485-5p, but not in the presence of anti-neg (Figure 2B). These results demonstrate that endogenous hepatic miR-485-5p is able to target the mutant APOA5 3′ UTR with the c.∗158C allele and decrease APOA5 3′ UTR luciferase expression. Our in silico studies primarily identified the APOA5 3′ UTR with the c.∗158C rare allele as a potential miR-485-5p target site. Our in vitro studies subsequently established that the APOA5 c.∗158C minor allele creates an illegitimate and functional miR-485-5p binding site. In the human liver, miR-485-5p might therefore downregulate mutant APOA5 at the posttranscriptional level, which could explain the strong GWAS-confirmed association between hypertriglyceridemia and APOA5∗2, which bears c.∗158C>T.3Pennacchio L.A. Olivier M. Hubacek J.A. 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This study clearly showed a close inverse association between apoAV and TGs in a cohort of 754 hypertriglyceridemic individuals (TGs > 150 mg/dl). Compared to the individuals with the g.4430 T/T major genotype, individuals with T/C and C/C genotypes displayed a 11% and 19% apoAV decrease, respectively, associated with a 9% and 18% TG increase, respectively.36Kim J.Y. Kim O.Y. Paik J.K. Lee S.H. Lee J.H. Association of apolipoprotein A-V concentration with apolipoprotein A5 gene -1131T>C polymorphism and fasting triglyceride levels.J. Clin. Lipidol. 2013; 7: 94-101Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar Several studies have suggested that APOA5∗2 might modulate APOA5 expression at the posttranscriptional level.20Hahne P. Krempler F. Schaap F.G. Soyal S.M. Höffinger H. Miller K. Oberkofler H. Strobl W. Patsch W. Determinants of plasma apolipoprotein A-V and APOA5 gene transcripts in humans.J. Intern. 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The functional interaction on in vitro gene expression of APOA5 SNPs, defining haplotype APOA52, and their paradoxical association with plasma triglyceride but not plasma apoAV levels.Biochim. Biophys. Acta. 2008; 1782: 447-452Crossref PubMed Scopus (36) Google Scholar, 25Ahituv N. Akiyama J. Chapman-Helleboid A. Fruchart J. Pennacchio L.A. In vivo characterization of human APOA5 haplotypes.Genomics. 2007; 90: 674-679Crossref PubMed Scopus (23) Google Scholar Our results support a miRNA posttranscriptional regulation of APOA5∗2. Consistent with our study, Palmen et al. performed a functional analysis of APOA5∗2 SNPs by using a luciferase reporter construct including part of APOA5∗2: the g.4430C>T, c.−3A>G, and c.∗158C>T rare variants. Palmen et al. reported approximately the same decrease in luciferase expression in HuH-7 cells as in our study. Although miRNA regulation was not suspected at that time, their findings are consistent with a targeting of APOA5∗2 by endogenous miR-485-5p expressed in this cell type. Additionally, Palmen et al. showed an equally reduced luciferase expression with the construct including only one APOA5 variant: the c.∗158C allele.24Palmen J. Smith A.J. Dorfmeister B. Putt W. Humphries S.E. Talmud P.J. The functional interaction on in vitro gene expression of APOA5 SNPs, defining haplotype APOA52, and their paradoxical association with plasma triglyceride but not plasma apoAV levels.Biochim. Biophys. Acta. 2008; 1782: 447-452Crossref PubMed Scopus (36) Google Scholar As in our results, these previous findings demonstrate that the c.∗158C>T rare variant alone provides luciferase modulation in this model. Additionally, the data presented in this study are in line with several previous studies that demonstrated that SNPs located either in miRNA genes or in mRNAs can affect miRNA-mRNA recognition and either abrogate or create miRNA binding sites.28Gong J. Tong Y. Zhang H.M. Wang K. Hu T. Shan G. Sun J. Guo A.Y. Genome-wide identification of SNPs in microRNA genes and the SNP effects on microRNA target binding and biogenesis.Hum. Mutat. 2012; 33: 254-263Crossref PubMed Scopus (295) Google Scholar This could account for diseases or phenotypic traits in various cellular pathways.29Richardson K. Louie-Gao Q. Arnett D.K. Parnell L.D. Lai C.Q. Davalos A. Fox C.S. Demissie S. Cupples L.A. Fernandez-Hernando C. Ordovas J.M. The PLIN4 variant rs8887 modulates obesity related phenotypes in humans through creation of a novel miR-522 seed site.PLoS ONE. 2011; 6: e17944Crossref PubMed Scopus (48) Google Scholar, 37Wynendaele J. Böhnke A. Leucci E. Nielsen S.J. Lambertz I. Hammer S. Sbrzesny N. 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Goldman S. et al.Identification of microRNAs as potential prognostic markers in ependymoma.PLoS ONE. 2011; 6: e25114Crossref PubMed Scopus (102) Google Scholar It has also been shown to be downregulated in ovarian epithelial tumors.43Kim T.H. Kim Y.K. Kwon Y. Heo J.H. Kang H. Kim G. An H.J. Deregulation of miR-519a, 153, and 485-5p and its clinicopathological relevance in ovarian epithelial tumours.Histopathology. 2010; 57: 734-743Crossref PubMed Scopus (110) Google Scholar miR-485-5p was not reported to affect lipid metabolism before our study. However, because APOA5 c.∗158C>T regulation would appear through the creation of an illegitimate target site, it is not obvious that miR-485-5p might physiologically regulate TG metabolism. Therefore, our work provides a comprehensive mechanism for the APOA5∗2 hypertriglyceridemic effect. We propose that, in humans, the miR-binding site created by the c.∗158C rare allele in the APOA5 3′ UTR causes liver posttranscriptional downregulation of APOA5 by miR-485-5p, a miRNA expressed in the human liver. This downregulation might at least partially account for the reported expression of APOA5∗2, its effect of rising plasma TG concentrations in humans, and the strong association between APOA5∗2 and both mild and severe hypertriglyceridemia. It remains to be clarified whether additional interactions with other APOA5∗2 SNPs are also required. The authors would like to acknowledge Michel Lagarde and Hubert Vidal for their helpful discussions, the nurses for blood drawing, and the Société Française d'Endocrinologie, Institut National de la Santé et de la Recherche Médicale, and Université Claude Bernard Lyon 1 for their support ("BQR accueil EC 2013" grants). S.R. received grants from the Fondation pour le Recherche Médicale, and P.M. received grants from the Fondation de France. Download .pdf (.47 MB) Help with pdf files Document S1. Figures S1 and S2 and Tables S1 and S2 The URLs for data presented herein are as follows:DIANA microT v.4, http://diana.imis.athena-innovation.gr/DianaTools/index.php?r=microtv4/indexmicroRNA.org, http://www.microrna.orgOnline Mendelian Inheritance in Man (OMIM), http://omim.orgPITA, http://genie.weizmann.ac.il/pubs/mir07/mir07_prediction.htmlRegRNA, http://regrna.mbc.nctu.edu.tw/TargetScan 6.2, http://www.targetscan.org/
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