Mechanism Study Of MiR-17-92 In Regulating Skeletal Muscle Cell Differentiation

Research background:Skeletal muscle is an important organ for maintaining the body's movements,and it is also the tissue with the most biological content,accounting for 40%of the human body.Skeletal muscle is susceptible to damage.Known external factors such as low temperature,puncture,inductance,and excessive exercise;or disease factors such as muscular dystrophy and neuropathy can cause damage to skeletal muscle.If not repaired in time,serious consequences such as dyskinesias,massive muscle loss,and even death can occur.The presence of muscle satellite cells contributes to skeletal muscle's self-repairing.After self-expanding,the satellite cells undergo myoblast differentiation and fuse to form new muscle fibers.Studies have shown that microRNAs play a key role in myogenic differentiation.The MicroRNA-17-92 family is a group of highly conserved and complex non-coding RNAs whose functions are involved in tumorigenesis,migration,and toxic effects.In recent years,some studies have shown that the miR-17-92 family is involved in the process of muscle myogenic differentiation.However,the specific roles and related mechanisms of each family member in this process have not been clear.In this study,C2C12 cells and MDSCs(primary bovine skeletal muscle-drived satellite cells)were used to text different microRNAs of the miR-17-92 family to explore their regulatory role and related mechanisms in myogenic differentiation.The specific conclusions are as follows:1.miR-17 and miR-20a promote myogenic differentiation of C2C12 and MDSCs cells.Cultured C2C12 and MDSCs cells were transfected with microRNA mimics of synthetic miR-17-92 family members.The effects of each member on myogenic differentiation were explored under differentiation conditions(DM)and proliferation conditions(GM).The results confirmed that miR-17 and miR-20a significantly promoted the differentiation of the two cells,while miR-18a,miR-19 and miR-92a had no significant effect on differentiation.In addition,miR-18a inhibitors can promote cell myoblast differentiation.2.miR-17 and miR-20a promote myogenic differentiation through the classical RISC silencing complex pathway.Using synthetic siRNAs to interfere with the two key proteins of RISC,AGO2 and GW 182,and performing myogenic differentiation experiments under the condition of GM,immunostaining results showed that transfection of miR-17 and miR-20a simultaneously transfected with siAG02 and siGW182 It can significantly inhibit the effect of differentiation and reduce the expression of MYHC protein.However,miR-17 and miR-20a alone can still promote differentiation.The results show that miR-17 and miR-20a promote myogenic differentiation through the classic microRNA-RISC silencing complex pathway.3.Screening and verification of miR-17 and miR-20a target genes.Ccnd2,Jakl,and Rhoc were selected as target genes downstream of miR-17 and miR-20a using RNA-seq sequencing technology combined with biological information analysis.The target genes were identified by real-time quantitative PCR,Western Blot,and dual luciferase reporting system.Furthermore,the effect of target genes on the differentiation promotion of C2C12 cells was verified under GM and DM conditions.The results showed that siCcnd2,siJakl and siRhoc can promote myoblast differentiation under DM condition,and siCcnd2,siJakl can promote effect under GM condition,but siRhoc effect is not significant.4.miR-19 inhibits the lethal effect of miR-17,and the combination of miR-17+19 further promotes myoblast differentiation.MiR-17 caused a lethal effect while promoting myoblast differentiation,but the addition of miR-19 suppressed the cell death caused by miR-17.Through transcriptome sequencing,it was found that miR-19 can target Pten,Socs3 and Tnfaip3,and activate AKT and ERK signaling pathways to inhibit apoptosis.At the same time,through transcriptome comparison and MYHC immunostaining tests,it was found that the combination of miR-19 and miR-17 further promoted the myogenic differentiation of C2C12.And promote the formation of myotubes by myocytes.5.miR-17+19 promoted the repair process of tibialis anterior muscle injury in mice.The artificially designed combination of shRNA-17 and shRNA-19 was made in virus,and concentrated by ultracentrifugation.Then injected into the tibialis anterior muscle of mice treated with Bacl2 in advance.Muscles were taken out on days 0,1,3,5,and 10,respectively,and stained with HE and immuno antibody.The results showed that compared with the blank control group,the microRNAs-treated group formed a larger number of new muscle fibers with a larger cross-sectional area,and shRNA-17+19 significantly promoted the process of muscle injury repair.In summary,this paper explores the regulatory effects of miR-17-92 family members on myogenic differentiation,and screens out the best pro-differentiation combination of miR-17+19.At the same time,the target gene mechanism of miR-17 to promote differentiation and miR-19 to promote survival was verified,and the ability of miR-17+19 to promote muscle damage repair was further explored,which provided new treatment strategies for diseases such as muscle damage and muscle atrophy.