Study On The Regulation Of MiRNA-101a In Goat Skeletal Muscle Satellite Cell Proliferation And Differentiation

MicroRNAs (miRNAs) involve in members of biological process, including early embryonic development, cell proliferation, differentiation and apoptosis, tumorigenesis and lipid metabolism. The related functional studies have confirmed that miRNA-101a (miR-101a) involved in the regulation of cell proliferation and differentiation, however, its role in skeletal muscle development was unclear.In this study, we amplified the sequence of precursor miR-101a adding its flanking sequence, analyzed the expression profile of miR-101a in different tissues and its role in the regulation of SMSC proliferation and differention. EZH2, the inhibitor of muscular cell differentiation, had the target site of miR-101a through dual luciferase report system. We further analyzed the change to the mRNA expression of miR-101a after overexpression or inhibition of miR-101a. We revealed that the molecular mechanism of miR-101a in the regulation of skeletal muscle development whereby the inhibition of its target gene EZH2. The main results are as follows:1. The precursor miR-101a adding its flanking sequence was 448 bp amplified from whole genome DNA of goat, the mature miR-101a in different species was highly conserved, the precursor miR-101a of goat conformed to the characteristics of microRNA structure on the online RNAfold software analysis, the relative expression of miR-101a in muscle tissues of triceps, longissimus dorsi, semimembranosus, and psoas major was higher than in other tissues, such as heart, liver, spleen, lung and kidney, by RT-qPCR and semi-quantitative PCR.2. During SMSC cultured in vitro, the mRNA expression of Pax7 was enriched in the proliferation stage, accompanied by the decreased expression during SMSC differentiation; MyoD had high expression in proliferation and the early stage of differentiation; MyoG had the highest expression level in the differentiation and fusion of 3 d; MyHC was increasedly expressed in the differentiation process. We detected the expression of miR-101a in 0 d、1 d、3 d、5d、7 d of SMSC differentiation by RT-qPCR, the results showed that the expression of miR-101a was up-regulated during SMSC differentiation.3. We induced SMSC overexpressed or inhibited of miR-101a to differentiation for 48 h before detection the mRNA expression changes of muscular cell marker gene Pax7、MyoD、MyoG、MyHC. The results indicated that expression of MyoG and MyHC was significantly up-regulated (P<0.01), but accompanied with the significant down-regulation of Pax7 and MyoD (P<0.01) after overexpressing the miR-101a; on the contrary, expression of MyoG (P<0.01) and MyHC (P<0.05) was significantly down-regulated but accompanied with the significant up-regulation of Pax7 (P<0.01) and MyoD (P<0.05) after inhibiting the miR-101a. Taken together, miR-101a can promote satellite cell differentiation.4. We applied the CCK-8 detection reagent to analyze the effect of miR-101a on the proliferation of SMSC, the results reported that miR-101a had no significant influence (p>0.05) on satellite cell proliferation compared with the NC.5. We used TargetScan, miRanda and PicTar application software to predict the target gene for miR-101a, then selected the candidate target gene EZH2 (inhibitor of muscular cell differentiation). The recombinant vector of PGL3-Control-EZH2 was successfully constructed. We cotransfected the miR-101a or inhibitor and recombinant vector plasmids into SMSC, then we verified the target sites of EZH2 3’-UTR region which complement to the seed region of miR-101 a through detection of luciferase activity.6. We detected the mRNA expression of target gene EZH2 during the SMSC differentiation of 0 d,1 d,3 d,5 d and 7 d adding after overexpressing or inhibiting of miR-101a, the results showed that it was down-regulated during SMSC differentiation however, the mRNA expression of EZH2 had no significant change (p>0.05) compared with the NC by RT-qPCR analysis after overexpression or inhibition of miR-101a. These results prompted that miR-101 a may promote skeletal muscle differentiation by inhibition the protein level of target gene EZH2.