| Embryonic stem cell (ESC), as a kind of pluripotent cell line, can be differentiated into nearly all sorts of cell types, including germ cells. Germ cells, as special adult cells in mammals, take the responsibility of transferring genetic materials to the next generation. By now, there are many researches on induction of mESCs into male germ cells, but with a low efficiency, the basic reason is that the regulating mechanism of male germ cell development in mammals is still unclear. Studies show that miRNA, as a kind of newly found small RNAs, might play an important role in spermatogenesis in mammals.In this study, several miRNAs, which might be related to gametogenesis, were initially selected and detected in all the mouse organs by semi- and real-time PCR to find a testis-specific miRNA. Specific miRNA mimics were synthesized and pri-miRNA-GFP plasmid vector was constructed, and they were transfected into mESCs. To study the effect on mESCs differentiation into male germ cells, specific miRNA was overexpressed, combined with retinoic acid (RA) induction, meanwhile. Genes targeted by the specific miRNA were then predicted by bioinformatics and dual-luciferase reporter vector was constructed. By miRNA mimics and vector co-transfection experiment, the predicted target gene was confirmed, and by this, the probable pathway regulated by this specific miRNA was initially studied.1. According to the published papers, several miRNAs (mmu-miR-34c, mmu-miR-449a, mmu-let-7e, mml-miR-122a), which might be related to gametogenesis, were initially chosen. The corresponding stem-loop primers were designed and these four miRNAs were detected in all the mouse organs by semi- and real-time PCR. Data showed that, in adult mouse testis, only miR-34c was specifically and highly expressed, with high but not specific expression of miR-449a; miR-122a was liver-specific and let-7e was widely expressed in nearly all the organs. Then the expression of miR-34c in mouse testes of different developmental stages and two sorts of testis cells was detected by real-time PCR. Data showed that, miR-34c was most highly expressed in testis of sexually matured mouse, exactly in spermatogenic cells; followed by mature mouse testis, with a little expression in testis from embryo to sexual maturity. There's no such phenomenon in mouse ovary.2. miR-34c mimics were symthesized by GenePharma, and pMiR-34c-GFP plasmid was constructed. it was overexpressed in mESCs, combined with 1μmol/L RA induction for 48 h, and the effect of miR-34c on induction efficiency of mESC differentiation into male germ cells was evaluated by cell morphology, Alkaline phosphatase (AP) staining, real-time PCR and immunofluorescence staining. Data showed that, after 48 h induction, compared with control, there were fewer AP positive clones and more spermatogonia-like cells in the transfected group, and the expression of Oct4 and c-Myc was down-regulated, with Vasa and Scp3 up-regulated. By this, we initially inferred that, miR-34c could improve the differentiation efficiency of mESCs into germ cells in vitro, and may play an important part during this process.3. Gametogenesis-related genes targeted by miR-34c were predicted by bioinformatics (such as RNA22, PicTar softwares), then RARg gene was selected and the recombined dual-luciferase reporter vector was constructed. By miRNA mimics and vector co-transfection experiment, the predicted target gene was confirmed, and by this, the probable pathway regulated by this specific miRNA was initially studied.In conclusion, we found a male germ cell specific miRNA--miR-34c, and data showed that it might be pivotal in mESCs differentiation into male germ cells in vitro; we also found a gene (RARg) targeted by miR-34c when it functions in regulating spermatogenesis, and proposed a mode hypothesis that miR-34c regulated male germ cell differentiation in mammals. |
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