The Mechanism Underlying The Effects Of MiR-22-3p On The Proliferation And Differentiation Of Primary Skeletal Muscle Cells In Hu Sheep

The meat production of Hu sheep affects the supply of mutton.How to improve the meat production is the primary focus of genetic breeding.The proliferation and differentiation of muscle cells and the fusion of mononuclear muscle cells to form multinucleated myotubes in the later stage are the basis of skeletal muscle growth and development.Clarifying the molecular mechanism of Hu sheep muscle cell development is expected to improve the meat yield in the molecular mechanism.Studies have shown that the growth and development of skeletal muscle requires a series of regulatory factors.MiRNA is a non-coding RNA with a length of about 22 nt,which can target and regulate the expression of mRNA and play an important role in the growth and development of muscle cells.MiR-22-3p is an important candidate molecule for regulating skeletal muscle development in Hu sheep.This research focuses on the regulation of gene expression by miR-22-3p.It preliminarily revealed the miR22-3p/IGFBP3/GNAI2 aixs play importmant roles in the proliferation and differentiation of primary skeletal muscle cells in Hu sheep.The main findings are as follows:（1）The effect of miR-22-3p on the proliferation and differentiation of skeletal muscle cells of Hu sheepThe miR-22-3p mimics and inhibitors were transfected into Hu sheep skeletal muscle cells.Through RT-qPCR,CCK-8,EdU and cell cycle experiments,we found that the proliferation marker genes PCNA,CDK2,Cyclin D1 in the mimic group were significantly lower than those in the control group（P<0.01）.The expression level,skeletal muscle cell viability,the number of EdU positive cells and the S phase of the cell cycle were significantly lower than those in the control group（P<0.01）.The expression levels of proliferation genes PCNA,CDK2 and Cyclin D1,the skeletal muscle cell viability,the number of EdU positive cells and the S phase of cell cycle in the transfection inhibitor group were significantly higher than those in the control group（P<0.01）.RT-qPCR and immunofluorescence results showed that after overexpression of miR-22-3p,the expression levels of cell differentiation genes MyOD and MyOG were significantly increased（P<0.01）,and the number of MyH3 positive myotubes increased.After inhibiting the expression of miR-22-3p,the expression levels of cell differentiation genes MyOD and MyOG were significantly decreased（P<0.01）,and the number of MyH3 positive myotubes decreased.The above results indicated that miR-22-3p inhibited the proliferation and promoted differentiation in skeletal muscle cells of Hu sheep.（2）miR-22-3p targets IGFBP3 to regulate the proliferation and differentiation of skeletal muscle cells of Hu sheepIGFBP3 was predicted to be a candidate target of miR-22-3p by mirDIP,miRTargets and RNAhybrid online bioinformatics analysis software.The dual-luciferase reporter system assay showed that the miR-22-3p mimic group significantly reduced the fluorescence activity（P<0.05）when compared with the control group.As an RNA-binding protein,IGFBP3 can improve RNA stability,and this gene is highly expressed in skeletal muscle cells of Hu sheep.After overexpression of IGFBP3,the expression levels of cell proliferation markers PCNA,CDK2,and Cyclin D1 were significantly higher than those in the control group（P<0.01）,and the cell viability was significantly or extremely significantly higher than that in control group（P<0.05 or P<0.01）,The number of EdU positive cells and the S phase of the cell cycle were significantly higher than those in control group（P<0.01）.After interfering with IGFBP3,the mRNA levels of cell proliferation genes PCNA,CDK2,and Cyclin D1 were significantly or extremely significantly lower than those in the control group（P<0.05 or P<0.01）.The cell viability was significantly or extremely significantly lower than that in the control group（P<0.05 or P<0.01）.The number of EdU positive cells and the cells in S phase were significantly lower than those in the control group（P<0.01）.The proliferation protein CDK2 was significantly lower than that in the control group（P<0.05）.RT-qPCR and immunofluorescence test results showed that the cell differentiation genes MyOD and MyOG were significantly or extremely significantly lower than those of the control group after overexpression of IGFBP3（P<0.05 or P<0.01）,and the number of MyH3 positive myotubes decreased.After inhibiting the expression of IGFBP3 gene,the expression levels of cell differentiation genes MyOD and MyOG were significantly increased（P<0.01）,and the number of MyH3 positive myotubes increased.It showed that there is a targeting relationship between IGFBP3 and miR-22-3p.IGFBP3 gene promotes the proliferation and inhibits differentiation in skeletal muscle cells of Hu sheep.（3）The effects of IGFBP3 downstream gene GNA12 on proliferation and differentiation of skeletal muscle cells of Hu sheepThe RNAct（Protein-mRNA interaction database）online software and RIP predicted that GNAI2 is a target gene of IGFBP3.After transfection of IGFBP3 interference fragment,the expression of GNAI2 gene was significantly decreased（P<0.01）.The mRNA levels of cell proliferation genes PCNA and CDK2 were significantly lower than those of the control group（P<0.01）.The cell viability was significantly lower than that of the control group（P<0.01）.The number of EdU positive cells was significantly lower than that in the control group（P<0.01）.After interfering with GNAI2 gene,the expression levels of cell differentiation genes MyOD and MyOG were significantly increased（P<0.05）,The number of MyH3 positive myotubes increased.It indicated that GNAI2 promotes the proliferation and inhibits differentiation in skeletal muscle cells of Hu sheep.Collectively,our preliminarily results suggested that the miR-22-3p/IGFBP3/GNAI2 aixs could play importmant roles in the proliferation and differentiation of primary skeletal muscle cells in Hu sheep.