Molecular Mechanism Of Mouse MSTN Knockout Myoblast Proliferation Restriction Based On Transcriptome And MiRNA Sequencing

Myostatin(MSTN)is a secreted protein mainly expressed in muscle tissue,which negatively regulates the proliferation and differentiation of myoblasts.In addition,it also controls the activation and proliferation of satellite cells(skeletal muscle stem cells),inhibits skeletal muscle growth and differentiation,and plays a key role in the growth and development of mammals.MSTN was first discovered in mice and highly conserved among different species.In animals such as humans,mice,cows or sheep,artificially induced disruption or natural mutations in MSTN can result in a broad increase in skeletal muscle mass,the "double muscle" phenotype.However,according to our previous studies,although the skeletal muscle mass was significantly increased after knocking out the MSTN gene,there was no infinite proliferation.Rhabdomyosarcoma(RMS)is a heterogeneous tumor found in human and domestic mammals,which develops due to genetic changes of mesenchymal progenitor cells/stem cells.RMS can also express some markers of normal skeletal muscle,such as N-cadherin,but it has unlimited proliferation ability.However,although the expression of MSTN decreased in both MSTN knockout animals and RMS patients,skeletal muscle quality of MSTN-KO animals increased significantly with no tumor cell-like infinite proliferation.Recent studies have shown that microRNAs(miRNAs or miRs)also play a key role in regulating the proliferation and differentiation of skeletal myoblasts through interaction with MSTN.Therefore,we suspect that the complete deletion of the most important negative regulator of skeletal muscle development and growth,MSTN,activates some miRNAs,some of which will target genes that regulate the growth and development of myoblasts,triggering a negative feedback mechanism to suppress excessive Skeletal muscle proliferation.In this study,we designed sgRNA on exon 3 of MSTN by CRISPR/Cas9.The expression vector was transfected into C2C12 by electroporation.Transcriptome and microRNA sequencing were performed on MSTN-KO and negative control cell lines.And transcriptome data of RMS was downloaded from the GEO database.We found that the complete loss of MSTN up-regulated 7 miRNAs targeting an interaction network consisting of 28 down-regulated genes,including TGFB1,FOS and RB1.By comparing the data of the MSTN-KO group and the WT group,it was found that two microRNAs were significantly increased in the MSTN-KO group,namely miR-130b-3p and miR-335-5p,and significantly decreased the expression levels of FOS and TGFB1 in MSTN-KO group were.Overexpression of miR-130b-3p and miR-335-5p directly inhibited FOS and TGFB1 and lead a decrease in the expression of FOS and TGFB1.Therefore,we knocked out the MSTN gene of mouse myoblasts by CRISPR/Cas9 and compared the sequencing data obtained by RNA-seq and miRNA-seq with the transcriptome data of RMS to find out the potential relationship between miRNA and muscle overgrowth.It is expected that these data will provide a theoretical basis for further exploration of the role of MSTN in the molecular mechanism of RMS.