MicroRNA-432Promotes Myoblast Proliferation And Affects Muscle Fiber Type Marker Gene Expression By Targeting Myoz1

MYOZ1encodes a kind of protein named calsarcin-2, a fast-twitch muscle exclusively expressed binding protein, who could bind to sarcomeric Z-disc and Z-disc associated proteins so that it could dedicate to stabilizeing the composition of muscle and relevant to the skeletal muscle Z-disc assembly. MYOZ1took part in the myocyte forging and two muscle fiber type formations. MYOZ1has a profound significance in skeletal muscle development, muscle fiber type switch as well as body image and meat condition in meat product industry. In this study we identified that MYOZ1was one of miR-432’s target genes, which may participate in the process of muscle fiber type proportion and that miR-432may affect myoblast proliferation. Our study further implicated that miR-432could work as a modulator contributing to the muscular development procedure. The results we have got as follows:(1)We examined the expression of MYOZ1in in several tissues (heart, liver, spleen, lung, kidney, small intestine, skeletal muscle and fat) from a Landrace pig, as well as in three developmental stages (embryonic period33day,65day and adult) of skeletal muscle by Q-PCR.(2) To further identify the association between miR-432and MYOZ1expression, we detected the mRNA expression level of in the myoblasts and myotubes which cultured in differentiation medium for Od,1d,2d,4d and6d of C2C12cells by Q-PCR.(3) To identify whether miR-432could bind to putative site of MYOZ1mRNA at3’UTR, we fused swine MYOZ1’s3’UTR to a duo-luciferase reporter gene plasmid and MYOZ1overexpress plasmid and induced into cells. Then we performed duo-luciferase assay, Western Blot and Immunofluorescence assay, we found that luciferase activity was significantly decreased when miR-432overexpressed, and the protein level declined with the miR-432.(4) To further investigate the biological function of miR-432in skeletal muscle development, we inhibited the endogenous miR-432using miR-432inhibitor in C2C12myoblast cultured in GM where this miRNA is expressed at high level. X-celligence analysis was conducted to estimate the proliferating abilities of cells by reading the cells index during their growth. It showed that the curve of miR-432inhibitor treatment was noticeable lower than the inhibitor N.C.(5) We induced the appropriate amount of miR-432duplex to the C2C12myoblast and maintained them in DM for collecting RNA on various time points (1day,3days and5days). We tested the two types of muscle fiber maker, MYH4and MYH7expression level. We found that the upregulation level of the slow muscle type marker MYH7was higher than that of MYH4. We overexpressed MYOZ1, and simultaneously knock down miR-432in C2C12myoblast and then cultured in DM for collecting RNAs on3time points (on1day,2days and4days). We detected the fast switch muscle maker, MYH4, and the slow switch muscle maker, MYH7on the indicated days of myoblast differentiation. The results showed that MYH4expression level raised more markedly than that of MYH7.