MiR-3031 Targets POU2F3 To Regulate Normal Goat Mammary Gland Development

MicroRNAs are small,highly conserved non-coding RNA molecules involved in the regulation of gene expression.miR-3031 is a functional miRNA and important mediator of many biological processes in the development of the mammary gland and lactation in dairy Goat.The purpose of this study was to investigate the relationship between POU2F3 and miR-3031 as well as their characteristic roles in goat mammary epithelial cells to provide a theoretical basis for the normal development of dairy goat mammary gland.The target gene of miR-3031 was verified by bioinformatics analysis,RT-qPCR,Western blot and dual luciferase assay.We acquired POU2F3 containing the binding sites of miR-3031 in its3’UTR,miR-3031 decreased the luciferase activity of PsiCHECK-POU2F3 3’UTR-wt indicating that POU2F3 is a direct target of miR-3031 through the binding to the POU2F33’UTR.The effect of miR-3031 on POU2F3 in goat mammary epithelial cells was elucidated by cell proliferation assay,accompanied by cell growth curve by using CCK-8 Kit assay,24 and 48 h post transfection.The main results are as follows: miR-3031 mimic’s transfection group significantly inhibited the luciferase activity of the wild-type vector.Real time PCR and Western blotting indicated that,the treatment of goat mammary epithelial cells with miR-3031 significantly decreases the mRNA and protein expression of POU2F3 levels compared to miRNA NC treated cells,the CCK8 kit was used to detect the proliferation of goat mammary epithelial cells.After 24 and 48 h,the result reveals that POU2F3 interference significantly promoted the proliferation of goat mammary epithelial cells.We use the siRNA technology for interfering the expression of POU2F3,synthesizes the siRNA mimic of POU2F3 gene(siPOU2F3),and transfects siPOU2F3 and si-NC into mammary epithelial cells.The results showed that POU2F3 was successfully interfered.At the same time,POU2F3 interference significantly inhibited the expression of Bcl-2 /Bax protein and significantly down regulated the expression of PI3 K protein.Subsequently,PTEN expression was extremely high after si-NC transfection.Taken together,miR-3031 inhibits the expression of the target gene POU2F3 in goat mammary epithelial cells;miR-3031 promotes the proliferation of mammary epithelial cells by the target gene POU2F3,by improving the phosphorylation of p-mTOR and mTOR,results downregulate the expression of mTOR in mammalian epithelial cells.