Ssc-MiR-21-5p and Ssc-MiR-615 Regulates the Proliferation and Apoptosis of Leydig Cells by Targeting SOX5

Leydig cells (LCs) are the predominant cells of androgen production, which plays key roles in spermatogenesis and maintaining male secondary sexual characteristics. Abnormal development of LCs affects androgen levels in vivo, affects fertility and may even lead to infertility. Little is known about the regulation mechanism on LCs' development and maturation in domestic animals, especially the regulation of non-coding RNAs. In this study, we continued to dig deeper in the previous RNA-seq data of porcine LCs from our group, combined with detecting the expression profiles in different tissues and different types of cells in the testis, to screen out candidate microRNAs (miRNAs) that may affect the regulation of LCs. A total of two miRNAs, ssc-miR-21-5p and ssc-miR-615 ("ssc" is omitted below), were finally determined. After overexpression and interference of miRNAs in vitro, the effects of candidate miRNAs on the proliferation and apoptosis of TM3 (mouse Leydig cell line) were explored. The results showed that miR-21-5p led to a decrease in TM3 cell density and p53 (apoptosis related protein) expression. Meanwhile, miR-21-5p decreased EdU positive cell numbers, but increased TUNEL positive cell numbers, suggesting miR-21-5p could inhibit proliferation and promote apoptosis. Conversely, miR-615 could increase TM3 cell density. Western blot and TUNEL assay indicated miR-615 inhibited apoptosis, but had no effect on proliferation. In addition, Sox5 was identified a potential target gene of these two miRNAs by Dual-Luciferase reporter system assay. Our findings about functions of miRNAs in TM3 and the mapping of miRNAs-target gene regulatory network would provide an important basis for the further elucidation of miRNAs in regulating pig LCs.