Characteristic Analysis Of Yak SIRT3 Gene And Its Effect On Cumulus Cells

Yak,as an important economic species in the plateau area,is the essential production material for the local people.However,its low fecundity limits the economic development of the plateau area.Cumulus cells,one of the main cells in the antral follicle,play an important role in regulating the development of ovary and oocyte.Therefore,the function of cumulus cells plays a vital role in improving the reproductive efficiency of mammals.Silent mating type information regulation2homolog-3(SIRT3),as a member of histone deacetylase family,mainly regulates cell metabolism and oxidative stress.At present,the research on SIRT3 mainly focuses on gene expression,metabolic regulation,cancer and aging.However,little is known about its function in cumulus cells.The purpose of this study was to investigate the effect of SIRT3 on yak cumulus cells and molecular regulation.The SIRT3 gene sequence of yak was cloned by RT-PCR and analyzed by bioinformatics.The relative expressions of SIRT3 in various tissues were detected by Quantitative Real-time PCR(qPCR);SIRT3 was silenced by RNA interference technology in cumulus cells.The relative expression levels of proliferation genes(PCNA),apoptosis genes(BAX,BCL-2,P53 and CASPASE3),antioxidant genes(IDH2,Mn SOD,CAT and SOD2)and progesterone synthesis-related genes(St AR,CYP11A1 and 3?HSD)were detected by qPCR.The results were shown as followings:(1)The open reading frame of SIRT3 gene was 1 002 bp,encoding 333 amino acids.SIRT3 was a basic,unstable hydrophilic protein without transmembrane structure and signal peptide by bioinformatics analysis.The homology results showed that SIRT3 was highly conserved in the evolution process.The results of tissue expression profile showed that the SIRT3 gene was widely expressed in various tissues of yak and it was highly expressed in spleen,testis,lung,ovary and muscle.(2)The culture system of yak cumulus cells was successfully established and SIRT3 was expressed in the cytoplasm and nucleus of cumulus cells,mainly in the nucleus.(3)SIRT3 was silenced by RNA interference technology in cumulus cells and the interference efficiency was 86%.After SIRT3 interference,the cumulus cell proliferation rate decreased significantly.ROS levels increased significantly.Progesterone levels decreased significantly.(4)After SIRT3 interference,the expression levels of BAX,P53 and CASPASE3 were significantly up-regulated.The expression levels of PCNA,St AR,CYP11A1 and3?HSD were significantly down-regulated.In conclusion,SIRT3 of yak was highly conserved in the process of evolution and widely expressed in various tissues;SIRT3 might significantly inhibit the proliferation and apoptosis,ROS production and progesterone secretion of yak cumulus cells by regulating the expression of related genes.This study provides basic data for further study of SIRT3 in the growth and development of yak and in the proliferation,apoptosis,oxidative stress and progesterone secretion of yak cumulus cells.It also provides the basis for studying the regulation of female reproduction and improving the production of yak.