Elucidation Of Mdfi Regulation Mechanism During C2C12 Cell Development

Muscle tissue is derived from embryonic mesoderm mesenchymal stem cells and belongs to soft tissue,which is an important part of animal body.Skeletal muscle is one of the most importantly metabolic organs in animals.It accounts for about 40% of the animal's body weight.In animal production,skeletal muscle development and protein deposition directly affect animal meat quality and production.The occurrence of muscle disease leads to severe muscle atrophy and weakness,which seriously affects the health of patients.The study of myoblasts growth and development is the basis for studying the growth and development of muscle tissue.Therefore,the study of the regulation mechanism of myoblast growth and muscle development process is very meaningful and urgently necessary in animal production and the optimization of therapeutic strategies for human diseases.During the process of muscle growth and development,it is regulated by various regulatory factors and signaling pathways.Myogenic regulatory factors(MRFs)is the most important regulator during muscle growth.Mdfi(Myod family inhibitor)is an inhibitor of myogenic differentiation(Myod)family members.During embryonic development,Mdfi is highly expressed in the sclerotome.Mdfi inhibits the transcriptional activation of Myod family transcription factors by blocking the nuclear localization signal and leading it to remain in the cytoplasm.Mdfi plays a broad and important regulatory role in kinds of tissue development and organogenesis.However,there are not many reports about Mdfi function in muscle development.The primary objective of this study was to investigate the role of Mdfi in the development of the myoblast C2C12 cell line.The Mdfi knockout and knock-in C2C12 cell model was constructed by CRISPR/Cas9 system.Combining phenotypic detection and with molecular experiment verification,the extensive and fine regulation function of Mdfi in myoblast proliferation,differentiation and migration was elucidated.The Mdfi promoter study revealed a regulatory loop for the transcriptional expression of the Mdfi gene by the upstream transcription factor.The molecular mechanism of Mdfi regulation of myoblast development was further explored by RNA-sequencing technology.The research results and innovations of this study will provide a new theoretical basis for study of myoblast development and skeletal muscle growth and development.The research results are as follows:1.We have successfully constructed Mdfi overexpressing and Mdfi-knockout C2C12 cell lines by CRISPR/Cas9 system,providing important research materials for subsequent research.2.The wild-type and Mdfi overexpressing C2C12 cells were analyzed by RNASequencing.The sequencing results showed that,in proliferative phase,there were 889 differentially expressed genes between the experimental group and the control group,including 608 up-regulating differentially expressed genes and 281 down-regulating differentially expressed genes.In the differentiation phase,there were 1522 differentially expressed genes in the experimental group and the control group,of which 434 were differentially up-regulated and 1088 were differentially down-regulated.GO analysis results showed that,in the proliferative phase,the differentially expressed genes were mainly enriched in Metal ion binding,Extracellular region,Intracellular,Extracellular space,Calcium ion binding,Cell adhesion and other GO terms.In the differentiation phase,the differentially expressed genes were mainly enriched in Membrane,Extracellular exosome,Extracellular region,Extracellular space,Extracellular space,Integral component of plasma membrane,Cell adhesion,Cell surface,Ion transport and other GO terms.KEGG signaling pathway analysis results showed that,in proliferative phase,the differentially expressed genes were mainly enriched in Pathways in cancer,Proteoglycans in cancer,Rap1 signaling pathway,Regulation of actin cytoskeleton,Adrenergic signaling in cardiomyocytes,c AMP signaling pathway,Calcium signaling pathway,and other signaling pathways.In differentiation phase,the differentially expressed genes were mainly enriched in Pathways in cancer,Focal adhesion,Cytokine-cytokine receptor interaction,Calcium signaling pathway,Axon guidance,c AMP signaling pathway,Rap1 signaling pathway,Cell adhesion molecules,ECM-receptor interaction,and other signaling pathways.These results showed that Mdfi overexpressing affects the differentiation process of C2C12 myoblast cells by changing the cell adhesion-related signaling pathway,signal transduction pathways,intracellular and extracellular matrix composition.3.During the proliferation of C2C12 cells,Mdfi overexpressing reduced the percentage of Edu positive C2C12 cells and Mdfi-knockout increased the percentage of Edu positive C2C12 cells.The flow cytometry results showed that Mdfi overexpressing significantly increased the number of cells in G1 phase,while significantly reduced the number of cells in the S and G2 phases.Mdfi-knockout Mdfi significantly reduced the number of cells in the G1 phase,while significantly increased the number of cells in the S and G2 phases.q RT-PCR results showed that Mdfi overexpressing significantly up-regulated the m RNA level of P21 and down-regulated the m RNA level of Ccnb1,Ccnd1,Ccne and Pcna.Mdfi-knockout significantly up-regulated the m RNA level of Ccnd1 and Pcna,while down-regulated the m RNA level of P21;Western blot results showed that Mdfi overexpressing decreased the protein level of Ccnb1 and Ccnd1,while increased the protein level of P21.In contrast,Mdfiknockout increased the protein level of Ccnb1 and Ccnd1,while had no effect on protein level of P21.Thus,Mdfi can up-regulate the expression of P21,down-regulate the expression of Ccnb1 and Ccnd1,inhibit the cell cycle of C2C12 cells transition from G1 to S and G2,and arresting in G1 phase,thereby inhibiting the proliferation of C2C12 cells.4.During the differentiation of C2C12 cells,Myosin immunofluorescence staining results showed that Mdfi overexpressing increased the percentage of Myosin positive cells in C2C12 cells,while Mdfi-knockout reduced the proportion of Myosin positive cells in C2C12 cells.q RT-PCR results showed that Mdfi overexpressing significantly up-regulated Myod,Myogenin,Myf5 and Myosin,Mdfi-knockout significantly down-regulated Myod,Myogenin,Myf5 and Myosin.Western blot results showed that Mdfi knockin increased protein level of Myod,Myogenin and Myosin,Mdfi-knockout decreased the protein level of Myod,Myogenin and Myosin.Thus,Mdfi can improve C2C12 cell differentiation through up-regulating Myod,Myogenin,Myosin expression.5.Mdfi overexpressing increased the number of Myhc I and Myhc IIa positive myotubes,while decreased the number of Myhc IIb positive myotubes.5.Mdfi overexpressing increased the number of mitochondria in C2C12 cell.Ch IP assay results showed that Mdfi can promote the expression of Myod,which enables Myod to bind to the promoter region of Camk2 b,increase its transcriptional activation.Also,Mdfi increased the expression of cell energy metabolism and mitochondrial oxidative phosphorylation related genes such as Pgc1?,Pdk4,Cs,Cox2,Cox4,Acadm,Acox1,Cycs and Atp5?1 which belong to downstream of Camk2 b to promote the conversion of the muscle fiber type from the glycolysis fast muscle fiber to the oxidized slow muscle fiber.6.During the migration of C2C12 cells,wound healing assay results showed that Mdfi overexpressing increased the migration ability of C2C12 cells,Mdfi-knockout decreased the migration ability of C2C12 cells.q RT-PCR results showed that Mdfi overexpressing significantly up-regulated the m RNA level of Fak And Paxillin,while Mdfi-knockout significantly down-regulate the m RNA level of Fak and Paxillin.In addition,Mdfi overexpressing increased the m RNA level of cell adhesion related genes,such as Cdh1,Cdh10,Col6a3,Itga4,Itgb4 and Itgb6,while,Mdfi knockout decreased the m RNA level of Col6a3,Itga4 and Itgb6.Western blot results showed that Mdfi overexpressing increased the protein level of p-Fak,Fak,p-Paxillin and Paxillin,while Mdfi-knockout decreased the protein of p-Paxillin and Paxillin.Ch IP assay and dual luciferase reporter system assay results showed that Myogenin can activate the transcriptional expression of Itga4 and Itgb4 by binding to the promoter regions of both Itga4 and Itgb4.Thus,Mdfi increased the transcription of Itga4 and Itgb4 by promoting the expression of Myogenin,and then promoted the adhesion and migration of C2C12 cells by further activating Fak-Paxillin signaling pathway.7.Mdfi gene promoter research results showed that the positive regulatory region of Mdfi promoter core was-1475/-1386 and +23/+120,and the core negative regulatory regions were-1306/-1084 and-291/-111.The comprehensive analysis results of dual luciferase reporter system,bioinformatics prediction and Ch IP assay showed that Myod was able to bind to the Mdfi promoter-1297/-1285 region and inhibited the transcriptional activity of Mdfi,while Myogenin was able to bind to the Mdfi promoter+29/+39 region and promoted the transcriptional activity of Mdfi.Co-immunoprecipitation assay results showed that Mdfi protein could directly interact with Myod and Myogenin,respectively.Thus,Mdfi protein can interact with both Myod and Myogenin.Meanwhile,there is indeed a binding site for Myod and Myogenin upstream of the Mdfi transcription initiation site.Myod could negative feedback regulate the expression of Mdfi and Myogenin could positive feedback to regulate the expression of Mdfi.In summary,this study clarified the function and molecular mechanism of Mdfi in the development of C2C12 cells by RNA-seq analysis and molecular experiments.In the proliferation process,Mdfi can inhibit the proliferation of C2C12 cells.In the differentiation process,Mdfi can promote the differentiation of C2C12 cells and promote the transformation of muscle fiber types from glycolysis type IIb muscle fibers to oxidized type I muscle fibers.In the migration process,Mdfi can promote the migration of C2C12 cells.Mdfi plays a broad and critical role in the myogenic development of C2C12 cells.