| MicroRNA(miRNA)is a type of endogenous,non-coding single-stranded small RNA with a length of about 20-24 nucleotides.Now,more and more miRNAs are found to play an important role in muscle development.Among them,some miRNAs are involved in the differentiation of myoblasts,such as miR-133 and miR-432;there are also some miRNAs that can specifically regulate the formation of muscle fiber types,such as miR-208 b and miR-499.However,there are still many miRNAs that have not been discovered in myogenesis.There have been many previous studies on MiR-129-5p,but most of them focused on tumors.At present,there is no report about miR-129-5p in skeletal muscle development.lncRNA acts as a ceRNA to adsorb miRNA and release its inhibitory effect on target genes,which is the main way for lncRNA to affect in vivo and in vitro functions.In addition,the previous sequencing of our laboratory showed that miR-129-5p is differentially expressed in the longest dorsal muscle and soleus muscles of large white pigs,indicating that miR-129-5p may be related to muscle development.Meanwhile,lnc Malat1 is a highly expressed non-coding RNA in skeletal muscle.Therefore,this experiment uses mouse C2C12 cells as a model to explore the role of miR-129-5p in the proliferation and differentiation of skeletal myoblasts and related upstream and downstream regulatory mechanisms.In this paper,C2C12 cells were used as materials to synthesize miR-129-5p mimics agomir and interfering agent antagomir respectively to increase and decrease the expression of miR-129-5p to study the effect of miR-129-5p in the process of myogenic proliferation and differentiation.Function;use online software to predict the downstream target genes of miR-129-5p and construct a dual fluorescein reporter vector for verification;construct a target gene overexpression vector for target gene recovery verification;finally,online software predict miR-129-5p binding LncRNA,To study the effect of the interaction between miR-129-5p and LncRNA on the process of skeletal muscle myogenesis.The main results obtained are as follows:1.MiR-129-5p has a limited effect on the myogenic proliferation of C2C12 cellsDuring the proliferation of C2C12 myoblasts,overexpression of miR-129-5p did not significantly affect the proliferation of cells.Ed U staining and CCK8 viability detection also did not reach a significant level;similarly,the cells were detected after interference with miR-129-5p.The related indicators of proliferation have not reached the significant level.2.miR-129-5p inhibits the myogenic differentiation of C2C12 cells and the expression of slow muscle-related genesIn the differentiation process of C2C12 myoblasts,after overexpression of miR-129-5p,MyHC immunofluorescence staining detects myotube fusion and differentiation index.The m RNA and protein levels of myogenic differentiation marker genes MyHC,Myo D,Myo G and slow muscle gene MyHC I are the same reduced significantly;on the contrary,after interference with miR-129-5p,the expression of myotube fusion index,differentiation index,myogenic related genes and slow muscle gene were significantly increased.3.Mef2a is the target gene of miR-129-5p in inhibiting the myogenic differentiation of C2C12 cellsThe online software predicted that the candidate target gene of miR-129-5p,Mef2a,was verified by the results of the dual luciferase system;Mef2a expression gradually increased during the differentiation process,while overexpression of miR-129-5p can reduce Mef2a expression;Overexpression of Mef2a can relieve the inhibitory effect of miR-129-5p on myogenic differentiation.4.The interaction between Malat1 and miR-129-5p regulates the myogenic differentiation of C2C12 cellsThe online website predicted that the upstream regulatory candidate Malat1 of miR-129-5p was obtained,and the dual luciferase reporter vector system verified this result;Malat1 promoted the myogenic differentiation of C2C12 cells,and the results of overexpression and interference were obtained the results;in addition,overexpression of LncMalat1 can promote myogenic differentiation by adsorbing miR-129-5p.In summary,miR-129-5p specifically targets Mef2a to inhibit C2C12 cell myogenic differentiation and slow muscle gene expression;also,Malat1 promotes C2C12 cell myogenic differentiation by sponging miR-129-5p. |
