Relationship Between SKIP And PI3K/Akt & P38 Signaling Pathways During C2C12 Myogenic Differentiation

Skeletal muscle fibers are the main components of skeletal muscle,and the number of muscle fibers is determined by the process of myogenic differentiation of muscle-derived cells before birth,which directly affects the growth potential and meat quality of livestock.Chromatin remodeling activation of MyoD-dependent muscle-specific genes is an important way to control myogenic differentiation,which is regulated by PI3K/Akt and p38 signaling pathway.SKIP(Skeletal muscle and Kidney enriched Inositol Phosphatase)is up-regulated in the process of myogenic differentiation and negatively regulates myogenic differentiation.The purpose of this study was to explore the relationship between SKIP and PI3K/Akt and p38 signaling pathways in C2C12 myogenic differentiation.The main results are as follows:1)C2C12 was induced to myogenic differentiation by 2% horse serum.SKIP expression was detected by fluorescence quantitative PCR and protein immunoblotting,DNA methylation status of SKIP regulatory region before and after myogenic differentiation was detected by bisulfite assay,open state of SKIP chromatin before and after myogenic differentiation was detected by ATAC-seq technique,binding of transcription factor MyoD and methylated histone H3K27me3 to SKIP promoter before and after myogenic differentiation was detected by ChIP.The results showed that the expression of SKIP was up-regulated in myogenic differentiation at both the level of protein and the level of mRNA;From that before myogenic differentiation,the distribution of DNA methylation in SKIP regulatory region changed after myogenic differentiation,but the degree did not differ significantly;Before myogenic differentiation,the chromatin in the promoter region of SKIP was closed or inhibited,and after differentiation,the SKIP chromatin was open;MyoD and SKIP promoters hardly bind before myogenic differentiation,but bind to SKIP promoters after myogenic differentiation;The binding level of H3K27me3 to SKIP promoter after myogenic differentiation was significantly lower than that before myogenic differentiation.2)C2C12 was induced to myogenic differentiation after overexpression of SKIP.Detections by fluorescence quantitative PCR,immunofluorescence and Western blot showed that the mRNA levels of differentiating marker genes such as Myogenin,MCK,MyoD and MEF2 C decreased,the protein levels of MyHC and Myogenin decreased,and the levels of p-Akt and p-p38 decreased.These results suggest that SKIP negatively regulates PI3K/Akt and p38 signaling pathways mediated myogenic differentiation.3)In the process of C2C12 differentiation induction,inhibitors LY294002 and SB203580 were used respectively.Detections by fluorescence quantitative PCR,immunofluorescence and ChIP showed that the mRNA levels of Myogenin,MCK,MyoD,MEF2 C,MyHC and SKIP were significantly or extremely significantly decreased after the treatment of inhibitors;the protein levels of MyHC and Myogenin were decreased;While PI3 K was inhibited by LY,SKIP promoter was hardly enriched with transcription factor MyoD,inhibited p38 with SB,the promoter of SKIP has the combination of MyoD.These results suggest that PI3K/Akt-mediated myogenic differentiation requires MyoD to bind to SKIP promoter,while p38-mediated myogenic differentiation may require MyoD to bind to SKIP promoter with other cofactors.The two pathways synergistically up-regulate the expression of SKIP.