Effect Of FHL3 Gene On Proliferation,differentiation And Muscle Fiber Types Of Bovine Skeletal Muscle Cells

Beef quality is one of the important factors to measure the efficiency of beef cattle breeding,and the process of muscle proliferation and differentiation and the type of muscle fiber types determine the growth and meat quality characteristics,so it is important to investigate the molecular regulatory processes affecting skeletal muscle development to improve beef quality.It has been shown that FHL3 gene is mainly expressed in skeletal muscle and differentially regulates the expression of MyHC isoforms in C2C12 cells,but whether it is involved in regulating the growth of bovine skeletal cells has rarely been investigated.Therefore,in this study,the FHL3 gene was used as a candidate gene and bovine skeletal muscle cells were used as a research model to investigate the effects of FHL3 gene on the proliferation and differentiation of bovine skeletal muscle cells,as well as on muscle fiber types,with the aim of elucidating the regulatory role of FHL3 gene in the process of bovine muscle development and muscle fiber formation,with the aim of increasing beef yield and improving meat quality in production practice.The main findings are as follows:1.Analysis of the expression pattern of FHL3 gene in different tissues and different differentiation periods in cattleThe full length of the CDS region of bovine FHL3,843 bp,was obtained by amplification,encoding a total of 280 amino acids.The results of real-time fluorescence quantitative PCR showed that FHL3 was expressed to different degrees in all tissues,with the highest expression in the longest dorsal muscle and the second highest expression in the lung compared to the heart tissue.In the process of induced differentiation of bovine skeletal muscle cells,the expression of FHL3 gradually increased with the increase of differentiation days compared with differentiation day 0,and reached the highest expression at differentiation day 6(P<0.01).2.Effect of FHL3 gene on proliferation,differentiation and myofiber type of bovine skeletal muscle cellsOverexpression of FHL3 in bovine skeletal muscle cells significantly upregulated the expression of CDK1,CDK2,and PCNA,which were markers of cell proliferation(P<0.05),and the opposite result was obtained after interference with FHL3(P<0.05);meanwhile,overexpression of FHL3 significantly downregulated the expression of Myf6,MyoD1,and MyoG,which were markers of cell differentiation(P<0.01),and the opposite result was obtained after interference with FHL3(P<0.01).CCK-8 results showed that FHL3 gene increased cell viability of bovine skeletal muscle cells to some extent,but its effect was not significant(P>0.05).The results of EdU assay showed that overexpression of FHL3 promoted the proliferation of bovine skeletal myoblasts and inhibited the proliferation of bovine skeletal myoblasts after interfering with FHL3.The results of cellular immunofluorescence showed that overexpression of FHL3 inhibited bovine skeletal myoblast differentiation,and interference with FHL3 promoted bovine skeletal myoblast differentiation;The results of cellular immunofluorescence showed that overexpression of FHL3 inhibited the differentiation of bovine skeletal muscle cells,and interference with FHL3 promoted the differentiation of bovine skeletal muscle cells;the results of real-time fluorescence quantitative PCR showed that overexpression of FHL3 upregulated the expression of MyHCⅡa and MyHC Ⅱx(P<0.05),downregulated the expression of MyHC Ⅰ(P<0.05),while it had no effect on the expression of MyHC Ⅱb(P>0.05),and Western blot showed that FHL3 promoted the expression of fast actin and inhibited the expression of slow actin,with the opposite result when FHL3 was inhibited.3.Effect of FHL3 gene on TGFβ1/Smad signaling pathwayOverexpression of FHL3 gene in bovine skeletal muscle cells inhibited the mRNA expression levels of Smad2 and Smad3 genes very significantly(P<0.01)compared with the control graph,but the effect on the mRNA expression of TGFβ1 and Smad4 was not significant(P>0.05),and the results were reversed when interfering.The protein expression of Smad2,Smad3 and Smad4 was significantly reduced by overexpression of FHL3,and the results were reversed when interfered,but FHL3 had no significant effect on the protein expression of TGFβ1(P>0.05).The phosphorylation levels of Smad2 and Smad3 proteins were significantly down-regulated when FHL3 was overexpressed(P<0.05)and highly significantly up-regulated when FHL3 was interfered with(P<0.01),suggesting that FHL3 may act through the TGFβ1/Smad pathway.The bovine FHL3 gene is involved in regulating the proliferation and differentiation of bovine skeletal muscle cells and muscle fiber types,but whether it acts through the TGFβ1/Smad pathway still needs further verification.The results of the study will provide some theoretical basis for the molecular breeding of beef cattle,and provide information for the in-depth study on the regulation of muscle development by FHL3.