MicroRNA-10b Regulates The Renewal Of Spermatogonial Stem Cells Through Kruppel-like Factor 4

MicroRNA plays an important role in cell proliferation,differentiation and apoptosis.It is an indispensable regulatory molecule thorough the whole development process.Spermatogonial stem cells self-renewal and spermatogenesis is a highly complex and sophisticated in which microRNA plays an extremely significant role in this process.So far,a large number of studies have been shown that differentially express microRNAs at different stages of the spermatogenesis process.We based on RNA-Seq data and referenced to some interrelated research's reports suggested that the ex pression of miR-10b significantly decline in the development of mice testis.We examined that the expression level of miR-10b in Thy1~+cells was 2.66 times higher than that Thy1~-cells.According to these hints illustrate miR-10b may have biological function in spermatogonia.First we respectively overexpressed,and knockdown miR-10b in vitro culture spermatogonia(cell line)originated from the CD-1 mouse.The consequences of EDU and CCK8 tests showed that overexpression of miR-10b could significantly promote the proliferation of SSCs,but had no significantly effect on the apoptosis of SSCs.However,the apoptosis rates of SSCs were significantly increased after knockdown miR-10b compared with control group.Second,the research reported that KLF4 is a direct acting target gene for miR-10b.KLF4 is a eukaryotic transcription factor involved in the regulation of cell proliferation,differentiation,and embryonic development.It is one of theimportant inducing factors for somatic cells reprogramming to induce pluripotent stem cells(iPS cells).In testes,especially after meiosis of germ cells and normal stertoli that KLF4 was high expression.it suggest that gene play an important role in the differentiation of testicular cells.We tested the expression of KLF4 in testes at the different phase of age founding that KLF4 increasing company with the age increase,but negatively correlated with mi R-10b expression.By software prediction,KLF4 is a direct acting target gene for miR-10b,and the interaction of miR-10b with KLF4 luciferase reporter assay has been confirmed in the interrelated research.In our study,overexpression of miR-10b in spermatogonia of CD-1 mouse lines significantly reduced the expression of KLF4,whereas knockdown miR-10b significantly increased the expression of KLF4.Furthermore,the test of EDU and CCK8 showed that knockdown KLF4 significantly promoted the proliferation of SSC_S in vitro.In conclusion,miR-10b interacts with the target gene KLF4 to regulate the proliferation and apoptosis of spermatogonia.This process is complex and sophisticated.It is helpful us to further understand the mechanism of SSCs self-renewal and differentiation,which is important for clinical diagnosis and treatment of male infertility.