MiR-208b Regulates Skeletal Muscle Fiber Types Conversion By Inhibiting Mettl8 Expression

Skeletal muscle is the main source of animal meat products,and its fundamental constituent unit is muscle fiber.Mature muscle fibers are divided into I,IIA,IIB,and IIX types according to the four myosin heavy chain isoforms?MyHC?:MyHC I?Myh7?,MyHC IIA?Myh2?,MyHC IIB?Myh4?and MyHC IIX?Myh1?,respectively.Different types of muscle fibers can be converted into each other.The previous research of our group showed that type I muscle fibers decreased and type IIB muscle fibers increased in skeletal muscle of MSTN?Myostatin?knockout Meishan pigs,while miR-208b expression was down-regulated and its target gene Mettl8?Methyltransferase like 8?was up-regulated,revealing that there is a certain connection.Since the seed sequence of miRNA is highly conserved,we took C2C12 cells and mice as the research objects,combined with molecular experiments,cell experiments and animal model construction,in order to explore the molecular mechanism of miR-208b regulates skeletal muscle fiber types conversion by targeting Mettl8.In this study,we first used RT-qPCR?Real-time quantitative polymerase chain reaction?and Western Blot?WB?to verify the previous sequencing results and explore the expression rules of miR-208b and Mettl8 in Meishan pigs.And then we induced myogenic differentiation of C2C12 cells to simulate the process of muscle fibers development and maturation.The transfection of miR-208b mimics and inhibitor in C2C12 cells was used to investigate the function of miR-208b in the expression of different muscle fiber types marker gene MyHC during differentiation.We also used bioinformatics analysis,dual luciferase experiments and molecular experiments to verify the targeting relationship of miR-208b and Mettl8,and investigated the impact of Mettl8 on the expression of My HC during differentiation using the interference experiment of Mettl8 in C2C12 cells.Finally,we constructed miR-208b and Mettl8 gene knockout mice models,and further explored the effects of miR-208b and Mettl8 on muscle fibers transformed through molecular experiments and HE staining.The following are detailed results.?1?Compared with the MSTN^+/+?wild group?,the expression of miR-208b and Mettl8 in MSTN^-/-?knockout group?were significantly down-regulated and up-regulated,respectively.MiR-208b is highly expressed in the longissimus muscle and heart of Meishan pigs at 65 days of embryonic period.And Mettl8 is widely expressed in various tissues of Meishan pigs.?2?During the process of myogenic differentiation,the change trend of miR-208b expression is consistent with the slow muscle fiber marker gene Myh7.After transfection of miR-208b mimics in C2C12 cells,its expression increased significantly.After 4 days of myogenic differentiation in C2C12cells with over-expressing miR-208b,the mRNA and protein levels of Myh4 and Myh7 were down-regulated and up-regulated,respectively,while the results were opposite after inhibiting miR-208b expression.The above results indicate that miR-208b can promote the conversion of fast muscle fiber to slow muscle fiber.?3?The binding site of miR-208b and Mettl8 3'UTR?Untranslated regions?was analyzed by bioinformatics analysis,and the targeting relationship between Mettl8 and miR-208b was verified by methods of dual luciferase experiment,RT-qPCR and WB.After transfection of Mettl8 small interfering RNA?siRNA?in C2C12 cells,it was found that the mRNA and protein expression of Mettl8 decreased significantly,the expression of Myh7 increased,while the expression of Myh4 decreased,indicating that Mettl8 could regulate the conversion of slow muscle fiber to fast muscle fiber.?4?The miR-208b and Mettl8 gene knockout mice models were successfully constructed,identified and bred.Compared with the wild type,the cross-sectional size of single muscle cell of the gastrocnemius muscle from the knockout mice have changed by HE staining of muscle fibers.RT-qPCR was used to detect the expression of MyHC and skeletal muscle energy metabolism-related genes in mice.The results showed that miR-208b and Mettl8 play opposite roles in the conversion of different types of skeletal muscle fibers,and can affect skeletal muscle energy metabolism by muscle fiber types conversion.In summary,miR-208b could regulate the transformation of different muscle fiber types by inhibiting the expression of Mettl8.