The Exploration Of The Regulatory Network Of Overexpression Mdfi On Proliferation And Differentiation Of C2C12 Cells Based On RNA-Seq

Muscle is an important part of the animal's body.The study of myoblasts is the basis for studying the growth and development of muscle tissue.During the process of muscle growth and development,it is regulated by various regulatory factors and signaling pathways.Mdfi(Myod family inhibitor)is an inhibitor of Myogenic regulatory factors(MRF).Mdfi plays a broad and important regulatory role in tissue development and organogenesis,but there are few reports on its regulation during muscle development.Our group successfully constructed a C2C12 cell line stably overexpressing Mdfi.It was verified by experiments that Mdfi can inhibit the proliferation of C2C12 cells,promote the differentiation of C2C12 cells,and found that Mdfi may lead to the transformation of muscle fiber types.In this study,cells of the proliferative phase and differentiation phase of C2C12 cells were selected for transcriptome sequencing,and differentially expressed miRNAs and genes were analyzed by bioinformatics methods to construct a network map of genes and miRNA-gene interactions.A regulatory network overexpressing Mdfi on mouse C2C12 cell proliferation and differentiation was fully revealed.The results of this study are as follows: 1.Overexpression of Mdfi in C2C12 cells proliferationAfter overexpression of Mdfi in the proliferative phase of C2C12 cells,889 differentially expressed genes,134 differentially expressed miRNAs,246 differentially expressed target genes,and 562 miRNA-gene interactions were obtained.A genetic interaction network map was constructed,and 28 core genes were obtained from the map;a miRNA-gene interaction network map was constructed,and 26 core miRNAs,16 core genes,and 44 core interaction pairs were obtained from the figure.2.Overexpression of Mdfi in C2C12 cells differentiationAfter overexpression of Mdfi in the differentiation phase of C2C12 cells,1522 differentially expressed genes,29 differentially expressed miRNAs,81 differentially expressed target genes,and 97 miRNA-gene interactions were obtained.A genetic interaction network map was constructed,and 29 core genes were obtained from the map;a miRNA-gene interaction network map was constructed,and 10 core miRNAs,6 core genes,and 13 core interaction pairs were obtained from the figure.3.Regulation of muscle fiber type transformation with overexpression of Mdfi in C2C12 cellsBy selecting the GO and KEGG entries related to muscle fiber type conversion reported in the differentiation stage,258 differentially expressed genes,29 differentially expressed miRNAs,34 differentially expressed target genes,and 51 miRNA-gene interaction pairs were obtained from the entries.A genetic interaction network map was constructed,and 17 core genes were obtained from the map;a miRNA-gene interaction network map was constructed,and 7 core miRNAs,8 core genes,and 12 core interaction pairs were obtained from the figure.4.Disease enrichment analysis of muscle-related miRNAsThe miRNAs in the proliferative and differentiation phases were introduced into the Human miRNA Related Disease Database(HMDD)database for enrichment analysis.The results showed that in the proliferative phase,7 core miRNAs were enriched into 5 human muscle diseases;in the differentiation phase,3 core miRNAs were enriched into 3 human muscle diseases.This analysis represents the enrichment relationship between core differentially expressed miRNAs after overexpression of Mdfi in mice C2C12 cells and miRNAs in human muscle diseases.5.Quantitative real-time PCR validationRandomly from the transcriptome sequencing results,some differentially expressed genes in the proliferative phase and the differentiation phase were selected for q RT-PCR.The results showed that the difference in the gene expression of the verified gene was consistent with the sequencing results.Similarly,miRNAs differentially expressed in the proliferative phase and the differentiation phase were selected from the transcriptome sequencing results for q RT-PCR.The results showed that 14 of the 16 miRNAs were consistent with the sequencing result,indicating that the sequencing results were good.In summary,we comprehensively explored the regulation of overexpression of Mdfi on the proliferation,differentiation and muscle fiber type transformation of mouse C2C12 cells from miRNA and gene association analysis,and analyzed the enrichment of core miRNAs in human disease database.The relationship was verified by q RT-PCR verification.Our analysis provides a reference for understanding the regulation of Mdfi on muscle growth and development.