The Preliminary Study Of MiRNA-194-5p Regulating Spermatogenesis Through Stra8 Gene

Objective:To explore whether miRNA-194-5p can regulate the process of spermatogenesis through Stra8.Methods:1.Stra8-knockout(Stra8-/-)and wild type(Stra8+/+)mice at the age of 1,2,3,4,5 and 8-weeks were collected and histologically analyzed.Testes of 11-day-old Stra8-/-and Stra8+/+ mice were used for miRNA Sequence.The expression level of differential expressed miRNAs in Stra8-/-and Stra8+/+ mice were verified by QRT-PCR.The expression level of differential expressed miRNAs in different tissues was also analyzed.2.PGL3-Stra8 3'UTR was constructed and then used to detect whether Stra8 can be directly regulated by miR-194-5p,miR-205-3p,and miR-3544-3p.3.Recombinant vector pMSCV-PIG-miR-194-5p was prepared and transfected into 293T cells to package the lentivirus.Then P19 cells were infected by the lentivirus and screened with puromycin.The expression of miR-194-5p in the cells was proved to be significantly increased by QRT-PCR.4.After induced by RA,the P19 cells were collected and used to detect the expression of Stra8 and Vasa.We also analyzed expression level of several proteins relevant with spermatogenic cells' proliferation and differentiation by Western blotting and cellular immunofluorescence.Results:1.There was no obvious morphological differences between 7-day-old Stra8-/-and Stra8+/+ mice.But the number of spermatogonia and spermatocytes in the seminiferous tubule of 14-day-old Stra8-/-mouse were decreased and showed abnormal hyperchromia.At the age of 3,4,5 and 8-weeks,few spermatogonia were detected in the seminiferous tubules,and some of the abnormal spermatogenic cells were gathered.2.We found six differentially expressed miRNAs between Stra8-/-and Stra8+/+ mice.The expression level of miR-194-5p,miR-205-3p,miR-3544-3p,miR-337-3p,miR-la-3p and miR-30c-1-3p in Stra8-/-and Stra8+/+ mice was verified by QRT-PCR.MiR-194-5p and miR-3544-3p were specially expressed in testis,while miR-205-3p was ubiquitously expressed in many organs in mouse.3.By using dual luciferase reporter gene assay,we found that Stra8-3'UTR can be negatively regulated by miR-194-5p,but not by miR-3544-3p and miR-205-3p.And mutant Stra8-3'UTR can't be regulated by miR-194-5p.4.PMSCV-PIG-miR-194-5p was successfully constructed.Lentivirus was also successfully produced.After infected by the lentivirus,the P19 cells can highly express miR-194-5p(P<0.05).5.Overexpression miR-194-5p in P19 cells can significantly reduce the expression level of Stra8 mRNA and protein.In addition,the expression of some proteins,which relevant with spermatogenic cells' proliferation and differentiation,such as Oct4,Plzf,Nanos3 and Vasa,were also changed significantly(P<0.05).Conclusions:In this study,we have found 6 miRNA differentially expressed in testes of 11-day-old Stra8-/-and Stra8+/+ mice.Among them,miR-194-5p can bind with 3'UTR region of Stra8 and negatively regulate the transcriptional activity of Stra8.Overexpression of miR-194-5p can decrease the expression of Stra8 mRNA and protein.And it can also regulate the expression of some proteins associated with proliferation and differentiation.So we speculated that Stra8 may be the direct target gene of miR-194-5p.miR-194-5p may regulate the process of spermatogenesis by direct or indirect targeted with Stra8.