title: MIR-34c regulates mouse embryonic stem cells differentiation into male germ-like cells through RARg.
abstract: Embryonic stem cells (ESCs) have the capacity to differentiate into nearly all sorts of cell types, including germ cells, which were regarded as one type of highly specialized cells in mammals, taking the responsibility of transferring genetic materials to the next generation. Studies on induction differentiation of murine embryonic stem cells (mESCs) into male germ cells, but with a low efficiency, basic reason is that the regulation mechanism of germ cell development in mammals is still unclear. miRNA might play an important role in spermatogenesis in mammals. In this study, several miRNAs, which might be related to spermatogenesis, were initially selected and detected in the mouse tissues by semi-polymerase chain reaction (PCR) and quantitative real time (qRT)-PCR to find a testis-specific miRNA. To study its effect on mESCs differentiation into male germ cells, miR-34c mimics were synthesized and pri-miR-34c-GFP plasmid was constructed, transfected into mESCs and combined with retinoic acid induction. The effects of miR-34c were analysed by morphology, alkaline phosphatase staining, qRT-PCR_and immunofluorescent staining. The results showed that miR-34c promoted mESCs differentiation into male germ-like cells, to some extent. Then miR-34c targeted genes were predicted by bioinformatics; Retinoic acid receptor gamma (RARg) was selected, and two dual-luciferase reporter vectors contained the normal and mutated 3'untranslated region of RARg were constructed, respectively. By miRNA mimics and vector co-transfection experiment, the predicted target gene-RARg was confirmed. In conclusion, we found a mammalian male germ cell specific miRNA--miR-34c, and it might be pivotal in mESCs differentiation into male germ cells through its target--RARg.
authors.affiliation: College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering and Technology, Key Lab for Animal Biotechnology of Agriculture Ministry of China, Northwest A&F University, Yangling, Shaanxi 712100, China.
authors.forename: Shanshan
authors.initials: S
authors.lastname: Zhang
authors.forename: Meng
authors.initials: M
authors.lastname: Yu
authors.forename: Chao
authors.initials: C
authors.lastname: Liu
authors.forename: Long
authors.initials: L
authors.lastname: Wang
authors.forename: Yue
authors.initials: Y
authors.lastname: Hu
authors.forename: Yaofu
authors.initials: Y
authors.lastname: Bai
authors.forename: Jinlian
authors.initials: J
authors.lastname: Hua
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D002454:Cell Differentiation / Q000235:genetics*; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008297:Male; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D009154:Mutation; D018168:Receptors, Retinoic Acid / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013094:Spermatozoa / Q000166:cytology / Q000378:metabolism; D014162:Transfection; D000097768:Retinoic Acid Receptor gamma
chemical_list: D020413:3' Untranslated Regions; D049452:Green Fluorescent Proteins; D035683:MicroRNAs; D018168:Receptors, Retinoic Acid; D000097768:Retinoic Acid Receptor gamma; C551848:MIRN34a microRNA, mouse