MiR-362 regulates the proliferation and apoptosis of porcine immature Sertoli cells through targeting the ZNF644 gene

Abstract

In testicular tissue, immature Sertoli cell proliferation ability determines the size of mature Sertoli cell populations, which further regulates the spermatogenesis in the adult male animals. Studies have demonstrated that microRNAs (miRNAs) participate in the regulation of the immature Sertoli cell proliferation and apoptosis, but the functions of most identified miRNAs remain unclear. In this study, based on previous RNA-seq results, we analyzed the regulatory role (s) of miR-362 in proliferation and apoptosis of porcine immature Sertoli cells. The ZFN644 gene was predicted to be a target gene of miR-362 using bioinformatics methods. The expression levels of miR-362 and ZNF644 gene were measured using qRT-PCR assay in developing porcine testicular tissues and in immature Sertoli cells transfected with either miR-362 mimic or miR-362 inhibitor. The dual luciferase reporter assay was used to determine the regulatory relationship between miR-362 and ZNF644. The results showed that a putative target site of miR-362 was located in the 3'UTR of ZNF644. The expression of miR-362 was significantly and negatively correlated with ZNF644 expression in the developing porcine testicular tissues. Co-transfection of miR-362 and psiCHECK2-ZNF644-WT 3'UTR luciferase vector significantly suppressed luciferase activity. The ZNF644 gene expression level was significantly regulated by miR-362, demonstrating that miR-362 targets ZNF644 gene and inhibits its expression in porcine immature Sertoli cells. Flow cytometry, CCK8, and EdU assays were used to measure the effects of over-expression of miR-362 or knockdown of ZNF644 on porcine immature Sertoli cell proliferation; Annexin V-FITC/PI staining assays and qRT-PCR technology were used to test the apoptosis and the expression levels of cell survival-related genes, respectively. Over-expression of miR-362 and knockdown of ZNF644 arrested the porcine immature Sertoli cells in G1 and G2 phases of the cell cycle, respectively, and inhibited proliferation, enhanced apoptosis in the porcine immature Sertoli cells, and significantly regulated the expression levels of cell survival-related genes. Taken together, these data indicate that miR-362 inhibits proliferation and promotes apoptosis in porcine immature Sertoli cells by targeting the ZNF644 gene, thereby providing the scientific basis for further study on the function(s) of miR-362 in the porcine spermatogenesis.