The Study On Mechanism Of Bta-miRNA-495 Affecting The Proliferation And Apoptosis Of Cattle Spermatogonial Stem Cells

The reproductive ability of male animals is closely related to testicular development and spermatogenesis.As an important part of testis,spermatogonial stem cells are the only adult stem cells in male animals that can transfer genetic material to their progeny,they have a strong dual ability of self-renewal and differentiation.They differentiate into spermatogonial and then undergo mitosis and meiosis to continuously produce sperm to transmit genetic information to their offspring.As a small non-coding RNA,the classic mode of action of micro RNA(miRNA)is to regulate related biological functions through targeted binding to the 3’-UTR region of mRNA.Relevant studies have shown that miRNA is widely involved in the spermatogenesis process and its importance in the development of male reproductive traits cannot be ignored.However,there are few studies on the function of miRNA in bovine spermatogonial stem cells.Therefore,in this study,based on the RNA-seq data of Angus bovine testis tissue in the early stage,bta-miR-495,which is differentially expressed in bovine testis tissues at different growth and development stages,was selected as the research object.After the expression characteristics of bta-miR-495 were analyzed,the effects of bta-miR-495 on the proliferation and apoptosis of bovine spermatogonial stem cells and its mechanism were systematically explored by using overexpression and interference methods,RT-qPCR,flow cytometry,EdU,CCK-8,dual luciferase gene reporter system,Western blot experiment and other techniques.This study provides a theoretical basis for understanding the molecular regulation mechanism of yellow cattle spermatogenesis and molecular selection of male reproductive traits.The main findings are as follows:1.Selection of bta-miR-495 and its effect on the proliferation and apoptosis of bovine spermatogonial stem cellsBased on the existing bovine testicular tissue transcriptome sequencing data,the differentially expressed bta-miR-495 was selected in the neonatal and after sexual maturity.The analysis of the expression characteristics of bta-miR-495 showed that the expression level in the neonatal bovine testis was significantly higher than that of adult cattle,which was consistent with the sequencing data.Taking the bovine spermatogonial immortalized stem cell line as a model,by overexpressing and inhibiting bta-miR-495,using RT-qPCR,flow cytometry,EdU,CCK-8 and Western blot experiments,it was shown that miR-495 inhibited the proliferation of bovine spermatogonial stem cells and promote apoptosis.2.Prediction and verification of bta-miR-495 target genesThe bioinformatics software Targetscan was used to predict the potential target genes of bta-miR-495,and the literature was reviewed to select three potential target genes(NABP1,CMTM8,RCN2)that may affect the proliferation and apoptosis of bovine spermatogonial stem cells.And through RT-qPCR,dual-luciferase gene reporter system to verify that RCN2 is the direct target gene of bta-miR-495.3.RCN2-ERK regulates the proliferation and apoptosis of bovine spermatogonial stem cells.In order to reveal the important role of RCN2,which is the target gene of bta-miR-495,in regulating the proliferation and apoptosis of bovine spermatogonial stem cells,we transfected RCN2 small interfering RNA into bovine spermatogonial stem cells.And we used RT-qPCR,flow cytometry,EdU,CCK-8 and Western Blot experiments to prove that specific knockdown of RCN2 leads to a significant decrease in the phosphorylation level of the key protein ERK of proliferation and apoptosis in the MAPK pathway,and bovine spermatogonial stem cells proliferation is inhibited,and apoptosis is promoted.These results suggest that bta-miR-495 and RCN2 play important roles in the process of proliferation and apoptosis of bovine spermatogonial stem cells,and further verify the targeted silencing effect of bta-miR-495 on RCN2.It provides help for further understanding of the complex molecular regulation mechanism in the process of spermatogenesis,and also provides a certain scientific theoretical basis for cattle breeding.