FoxO1对牛骨骼肌细胞增殖、凋亡和分化的调控OA北大核心CSTPCD

[Objective]Skeletal muscle is an important component of animal body,and its growth and development directly affect the meat yield of livestock and poultry.As an important transcription regulator,fork head box protein O1(FoxO1)is closely related to the growth and development of skeletal muscle.Exploring the effects of overexpression of FoxO1 gene on the proliferation,apoptosis and differentiation of bovine skeletal muscle cells could provide the theoretical basis for genetic improvement of beef.[Method]The tissue expression profile of FoxO1 was constructed by real-time fluorescence quantitative PCR(qPCR).To test the cell differentiation performance of bovine skeletal muscle cells which were isolated by enzyme digestion,the myotubes formation was observed and the expressions of differentiation marker genes were detected by qPCR.The subcellular localization of bovine skeletal muscle cells was carried out by immunofluorescence technique.In order to improve the expression of FoxO1 in bovine skeletal muscle cells,bovine FoxO1 overexpression adenovirus were designed and packaged.The effect of FoxO1 overexpression on the relative proliferation rate of cells was detected by EdU staining.Flow cytometry was used to detect the effect of FoxO1 overexpression on cell cycle distribution.The effects of FoxO1 overexpression on the expression levels of genes related to proliferation,apoptosis and differentiation of bovine skeletal muscle cells were investigated by qPCR.[Result]The results of tissue expression spectrum showed that FoxO1 was expressed in many tissues,with the highest expression in the back fat of adult cattle and the lowest expression in the longissimus dorsi muscle tissue,while the expression of FoxO1 in the longissimus dorsi muscle tissue of calves was significantly higher than that of adult cattle(P＜0.01).The results of subcellular localization showed that FoxO1 was expressed in the nucleus and cytoplasm of bovine skeletal muscle cells,and the fluorescence intensity in the nucleus was higher than that in the cytoplasm.The FoxO1 overexpression vector was successfully constructed,and the packaging and propagation of FoxO1 recombinant adenovirus were completed.After infecting bovine skeletal muscle cells,the FoxO1 expression level was significantly improved(P＜0.01).EdU detection showed that overexpression of FoxO1 significantly reduced the cell proliferation rate(P＜0.01),and flow cytometry showed that overexpression of FoxO1 significantly increased the number of cells in G1 phase and decreased the number of cells in S phase and G2 phase,inhibited the transformation of cells in G1/S phase and reduced the formation of cells in G2 phase.Further detection by qPCR found that the proliferation-related genes of PCNA,CDK1,CDK2,CCNA2,CCNB1,CCND1 and CCNE2 were significantly down-regulated(P＜0.01),the apoptosis-related genes BAD and BAX were significantly up-regulated,and the apoptosis-inhibiting gene BCL2 was significantly down-regulated(P＜0.05).After FoxO1 was overexpressed,the myotube formation of bovine skeletal muscle cells decreased,and the expression levels of MYOD,MYOG,MYF5,MYF6 and MYHC related to skeletal muscle differentiation were significantly decreased by qPCR detection(P＜0.05).[Conclusion]FoxO1 was expressed in different tissues of cattle,and it was a ubiquitous transcription regulator.There were differences in expression at different stages of the growth and development of longissimus dorsi muscle,which played a role in stage regulation.FoxO1 played a transcriptional regulatory role in both the nucleus and cytoplasm,especially in the nucleus.Overexpression of FoxO1 might inhibit the proliferation and differentiation of bovine skeletal muscle cells by inhibiting the expression of genes related to cell proliferation and muscle cell differentiation,and might promote the apoptosis of bovine skeletal muscle cells by up-regulating the expression of apoptosis-promoting genes and down-regulating the expression of apoptosis-inhibiting genes.