MiR-322 Regulates Spermatogonial Stem Cells Self-renwal And Differentiation Through Rassf8

Spermatogenesis is an extremely complex process that originates from spermatogonial stem cells(SSCs)self-renewal and differentiation and is regulated by a variety of endocrine and paracrine signals.Many studies have shown that microRNAs are widely expressed in mouse testis,and lots of microRNAs play important roles in spermatogenesis,especially the development of SSCs.The purpose of this study was to investigate the mechanism of miR-322 regulating SSCs self-renewal and differentiation in vitro.The expression of miR-322 in SSCs,pachytene spermatocytes,round spermatids and mature sperm was detected by qRT-PCR,and it was found that the expression of miR-322 gradually decreased with the development of SSCs.Then,miR-322 overexpressing lentivirus vectors,miR-322 inhibition lentivirus vectors and their respective control lentivirus vectors were respectively infected with SSCs in vitro,and proliferation experiments such as CCK8 and EdU showed that overexpression of miR-322 can promote SSCs proliferate and up-regulate the expression of stemness genes,such as Gfrα1,Etv5 and Plzf,while inhibition of miR-322 inhibits the proliferation of SSCs and up-regulates the expression of markers associated with differentiation,such as Stra8,C-kit and Bcl6,and the structure of synaptic complex can be detected by SYCP3 immunoassay.Next,in order to explore the mechanism how miR-322 regulates SSCs self-renewal and differentiation,we used TargetScan to predict target genes and predicted that miR-322 could bind to Rassf8 3’UTR.Experiments such as qRT-PCR,western blotting and dual luciferase reporter assay proved that Rassf8 is a target gene of miR-322.MiR-322 can promote SSCs proliferat in vitro via Rassf8.Subsequently,we constructed Rassf8 overexpressing lentivirus vectors,Rassf8 inhibition lentivirus vectors,and their respective control lentivirus vectors,and then we used them to infect SSCs.The results of CCK8 and EdU showed that the overexpression of Rassf8 inhibited the proliferation of SSCs in vitro,while the inhibition of Rassf8 promoted the proliferation of SSCs.Finally,we treated SSCs with lithium chloride,the activator of WNT/β-catenin pathway,and detected the expression levels of miR-322 and Rassf8 in these SSCs.It was found that miR-322 expression was increased while Rassf8 expression was decreased.Then we detected the expressionof downstream genes of SSCs infected with miR-322 inhibition lentivirus vectors or Rassf8 inhibition lentivirus vectors,we found that inhibition of miR-322 leads to down-regulation of downstream gene expression,while inhibition of Rassf8 causes up-regulation of downstream genes,we also used western bloting to detect the protein expression of downstream genes.Finally,the cell cycle of each group of SSCs was detected.It was found that overexpression of miR-322 increased the ratio of S phase and G2/M phase,while inhibition of miR-322 caused cell arrest in G0 /G1 phase,these results indicated that miR-322 can regulate SSCs self-renewal and differentiation through the WNT/β-catenin signaling pathway.In conclusion,our results indicate that miR-322 can regulate SSCs self-renewal and differentiation via Rassf8 in vitro.Our research not only provides a noval mechanism for miRNAs regulating SSCs,but also provides a theoretical basis for the diagnosis,treatment and prevention of male infertility.