Study On Overexpression STAR Gene Affects The Proliferation And Differentiation Of Bovine Myoblasts And Precursor Adipocytes

Beef is the third most consumed meat in the world.With the upgrading of beef consumption structure,high-quality and high-grade beef is becoming more and more popular among consumers.To improve the yield and quality of beef is the focus of cattle feeding and breeding.Marbled beef is fat tissue surrounding the muscle fibers in the muscle of beef cattle.The more intramuscular fat deposits,the more obvious the pattern,and the grade of beef will rise accordingly.At the cellular level,muscle cells and fat cells are intermingled,and the two cells may regulate each other through autocrine or paracrine that ultimately affecting the quality of beef.In order to reveal the molecular mechanism of the dialogue between fat and muscle tissue and to explain the principle of marbled beef formation,it is necessary to identify the key genes that regulate the differentiation and proliferation of both bovine adipocytes and myoblasts.Steroidogenic acute regulatory protein（STAR）is a differentially expressed gene screened by transcriptomic sequencing during the mutual regulation of preadipocyte and myoblast.This gene is a star factor in the endocrine field,but its biological functions such as skeletal muscle growth and development and cell proliferation and differentiation are rarely studied.Therefore,STAR was selected as the target gene in this study.Real-time quantitative PCR was used to map the spatio-temporal expression of STAR gene in Qinchuan cattle,and the expression characteristics in bovine myoblasts and precursor adipocytes.In addition,We detected the influence of STAR gene on proliferation and differentiation of bovine myoblasts and adipocytes at both the cellular and molecular level by using the adenovirus mediated gene over-expression biotechnology.The main results are as follows:1.The relative expression of STAR gene was low in bovine adipose tissue and muscle tissue,and increased during the adipocytes differentiation.The results showed that STAR gene was expressed in low abundance in adipose tissue and muscle tissue.The expression level of STAR gene in muscle tissue of newborn cattle was significantly lower than that of adult cattle,while the expression level in adipose tissue of newborn cattle was extremely significantly higher than that of adult cattle.In the process of bovine myoblast differentiation,the expression of STAR gene firstly increased and then decreased,reaching the peak on the 4^th day.In the differentiation process of bovine adipocytes,the expression of STAR gene decreased on 2^nd day and continued to increase.2.Overexpression STAR inhibited the proliferation of bovine myoblasts and the expressions of PCNA;also inhibited the differentiation of bovine myoblasts and the expression of MYOG and MYH3.The results of RT-q PCR,western-blot,CCK8,Ed U staining and flow cytometry showed that when STAR gene was overexpressed,the expressions of cell cycle genes MCM3,MCM6,PCNA,CCNA2,CCND at m RNA level and PCNA at protein level were significantly inhibited（P<0.01）,and the expression of cell cycle suppressor gene P21 at m RNA level was significantly promoted（P<0.01）.The proliferative activity of myoblasts was inhibited and remained in the G0/G1 phase.The results of cell observation,RT-q PCR,western-blot showed that when STAR gene was overexpressed,the formation of muscle tubes was inhibited.The expression of myogenic genes MYOG,MYOD,MYH3,My F5 and MRF4 at m RNA level and MYOG,MYH3 at protein level were significantly inhibited（P<0.01）.In addition,lipids were accumulated in myoblasts,and the m RNA expressions of lipid-related genes FABP4,LPL,DGAT,PPARγ,FAS,SREBP1 and LPIN1 were significantly promoted（P<0.01）.3.Overexpression STAR inhibited the proliferation of bovine adipocytes and the expression of PCNA;And inhibited the differentiation of bovine adipocytes and the expression of C/EBP-α.The results of RT-q PCR,western blot,CCK8,Ed U staining and flow cytometry showed that when STAR gene was overexpressed,The expressions of cell cycle genes MCM3,PCNA and CCNA at m RNA level and PCNA,CCNB and CCND at protein level were significantly inhibited（P<0.01）,and the expressions of cell cycle suppressor genes P21 and P27 at m RNA level were significantly promoted（P<0.01）.The proliferative activity of adipocytes was inhibited and remained in the G0/G1 phase.The results of oil red O staining,triglyceride detection,RT-q PCR,western blot showed that when STAR gene was overexpressed,the formation of lipid droplet and synthesis of triglyceride were inhibited,and the expression of adipogenesis related gene C/EBPαat m RNA and protein levels was significantly inhibited（P<0.01）.In conclusion,overexpression of STAR gene inhibits the proliferation and differentiation of bovine myoblasts and adipocytes.This co-regulatory effect suggests that STAR is involved in the molecular dialogue of adipose-muscle tissue and can be used as a candidate gene for molecular breeding of beef.