Analysis Of Myostatin Dysfunction Affect Myoblast Cells Development Regulation And Mitochondrial Function Based On Omics Technology

Backgroud Myostatin,a negative regulator of skeletal muscle mass,also known as GDF-8,which is a secreted growth factor of the transforming growth factor-beta(TGF-?)superfamily.MSTN is a highly conserved negative regulator that regulates muscle development and growth by controlling proliferation of muscle precursor cells.MSTN inactivation leads to impressive muscle hypertrophy,causing double muscling phenotype in livestock species.At present,most studies on the molecular mechanism of MSTN regulating muscle development have focused on the traditional methods,and there have been a few reports on the molecular mechanism of transcriptome and proteomics combined analysis of MSTN gene regulating muscle development.Sheep as an important economic animal in the field of agricultural production,the improvement of its yield and quality has never stopped.The rapid development of genome editing,transcriptome and proteomics is conducive to screening out a large number of proteins and signal pathways related to muscle development from mammalian cells and animals with MSTN gene knockout.Aims Here,CRISPR/Cas9 technology was used to establish MSTN gene knockout C2C12 monoclonal cells and the laboratory existing muscle tissue samples of northern Shaanxi white cashmere goats with MSTN gene knockout.Through transcriptomics and proteomics analysis,potential regulatory factors and signal pathways related to muscle development were screened out.By up-regulating or down-regulating these regulatory factors in C2C12 cells,the function of regulators was studied to clarify the relevant mechanism of MSTN gene regulate muscle development.Finally,based on the results of cytological experiments,their functions and their possible mechanisms in the proliferation and differentiation of skeletal muscle cells were confirmed,providing new ideas for animal breeding.Methods 1.Mouse myoblast C2C12 was used as the research model,and CRISPR/Cas9 technology was used to construct a mouse myoblast cell clone accompany with MSTN gene knockout.Mutation efficiency was detected by T7 EI,and mutation types were analyzed by Sanger sequencing.m RNA and protein expression levels of MSTN were detected by RT-q PCR and Western blot.2.Analysis of MSTN in the C2C12 cell clone based on Proteomic: the experiment was divided into control group(empty vector group)and treatment group(MSTN gene knockout group).Samples of mouse myoblast C2C12 cells were collected and total protein,protein quantification,SDS-PAGE,protein enzymatic hydrolysis and LC-MS /MS were analyzed.The protein sequence database was selected for bioinformatics analysis.By analyzing and comparing the differences of protein expression profiles between the two groups,and combining with the related literature reading,in order to found that some of the differentially expressed proteins that can regulate the proliferation and differentiation of muscle cells and revelant the signaling pathways.3.Based on the proteomics of cells reserch.Transcriptome analysis of the muscle tissue of northern shaanxi white cashmere goat with MSTN gene knockout: the experiment was divided into three groups: fibroblast growth factor 5(FGF5+/-)gene knockout alone(FGF5+/-),MSTN and FGF5 gene knockout simultaneously(FM+/-)and control group(WT).High-throughput RNA-Seq technology was used to determined gene change.Bioinformatics analysis was used to screen the differentially expressed genes and signaling pathway of MSTN gene knockout between northern Shaanxi white cashmere goat and wild type.4.Comprehensively analyze the results of proteomics and transcriptome to seek common differentially expressed proteins and molecular signaling pathways.5.MSTN gene knockout and Lentivirus-mediated overexpression of FST in myoblasts and the corresponding control group: the m RNA and protein expression levels of MSTN,vital differential proteins in signaling pathways associated with muscle development were detected by RT-q PCR and Western blot.To verify the action mechanism of MSTN gene on muscle development regulation.To further explore the BNIP2-CDC42 mediated p38 MAPK signaling pathway to promote the differentiation of myoblast cells.After myostatin deletion,BNIP2-CDC42 was determined to activate the p38 MAPK signaling pathway by Western blot and immunoprecipitation.6.Based on animal welfare factors,the effects of MSTN gene knockout on mitochondrial biogenesis and energy metabolism were studied.Mitochondrial markers PGC-1?,Cox1,Cox2,ND1 and ND2 were detected by RT-q PCR and Western Blot.Cell Counting kit-8(CCK-8)Cell proliferation,Mito Tracker ? Red CMXRos and Mito Tracker Green staining myoblast mitochondria,glucose uptake,ATP content,mitochondrial membrane potential,lactic acid concentration,RT-q PCR,and Western blot assay were performed.Results 1.A total of 3003 proteins were identified and quantified in KO cells.Among these proteins,77 proteins were significantly differentially expressed with p value < 0.05 and ? 2,among differentially expressed proteins 38 upregulated and 39 downregulated,in MSTN KO C2C12 cells.These significantly altered proteins are involved in mitochondrial localization,cellular lipid metabolism,fatty acid synthesis,cell cycle,and ubiquitination of regulatory proteins.2.The differential expression profile of muscle tissue transcriptome of northern Shaanxi white cashmere goats showed that the significantly differentially expressed genes were mainly enriched in fatty acid synthesis,calcium ion signaling pathway,c AMP signaling pathway,MAPK signaling pathway and cell adhesion molecule(CAMs)after MSTN gene knockout.3.Analyzed proteomic and transcriptome data and combined literature review showed that MSTN gene knockout by BNIP2-CDC42 mediated p38 MAPK signaling pathway can regulate muscle cell development.4.p38 MAPK inhibitors were added to cells of both treatment group and control group after MSTN gene knockout,and related-genes and proteins of the p38 MAPK signaling pathway were significantly reduced by analyzed RT-q PCR and Western blot.It was proved that the inactivation of MSTN gene can activate the BNIP2-CDC42-p38 MAPK signaling pathway in skeletal muscle myoblast cells,thus promoting cell differentiation.5.Myostatin can affect the mitochondrial biogenesis and energy metabolism of C2C12 cells in skeletal muscle.Conclusion Our study showed that MSTN gene of C2C12 in skeletal muscle myoblasts of mice can be successfully knocked out by CRISPR/Cas9 gene editing technology.The CDC42-BNIP2-KIF5 B protein complex was selected to promote the differentiation of muscle cells by activating the p38 MAPK signaling pathway through the combined analysis of proteomics and transcriptome.At the same time,MSTN dysfunction can promote the mitochondrial biogenesis of myoblasts,but can not influence cells vitality.