Role Of MyHC ⅡA/X-AS In Skeletal Muscle Differentiation And Muscle Fibertype Composition In Pigs

Meat is one of the major animal products,mainly composed of different types of myofibers,and the composition of myofibers types determines meat quality.Myofiber type composition is regulated by many genetic factors,and long noncoding RNA(lncRNA)is an important one.LncRNAs are RNA of over 200 nucleotides with no coding potential,which play crucial regulatory roles in the process of life.In this study,the MyHC IIA/X-AS was cloned and identified as a natural reverse transcript of myosin heavy chain(MyHC)locus in pigs,and its regulation of pig muscle production was explored.In this paper,the full length of MyHC IIA/X-AS lncRNA was cloned by biological software and RACE,etc.;Then the expression of lncRNA in different tissues of Guanzhong black pig,subcellular localization and the correlation gene of expression of locus-related genes were detected by qPCR;Subsequently,the role of MyHC IIA/X-AS on proliferation and differentiation of myocytes were investigated.Finally,the lncRNA was explored to regulate the composition of different muscle fiber types by interacting with miRNA-130 b and MyHC IIx.The main results are as follows:1.MyHC IIA/X-AS lncRNA was cloned by RT-PCR and online blast tools shown it is located on chromosome 12 of pig,and its size is about 4~5kb.The expression level of MyHC IIA/X-AS lncRNA in different tissues of pigs is detected by qPCR.The lncRNA is higher enriched in muscle tissue.The results of nuclear separation experiment and fluorescence in situ hybridization(FISH)showed lncRNA was mainly found in cytoplasm of porcine skeletal muscle;The results of half detection analysis showed the stability of MyHC IIx and MyHC IIA/X-AS lncRNA was higher than that of MyHC IIx;MyHC IIA/X-AS lncRNA was gradually increased during myogenic differentiation,and the same pattern was also found during the postnatal growth of piglets.Correlation analysis show that the expression pattern of MyHC IIA / X-AS lncRNA is more similar with that of MyHC IIx.2.In isolated and purified porcine satellite cells,siRNA inhibited MyHC IIA/X-AS the expression of genes related with cell proliferation Cyclin E,Cyclin D and PCNA were significantly up-regulated compared with the control group(P<0.05)and cell viability was also significantly increased.The number of Edu-positive cells was also significantly increased(P<0.05).The flow cytometry results shown that the percentage of S-phase cells was significantly higher than that in the control group(P<0.05).In addition,the expression of myogentic differentiation-related genes MyHC,MyoG and MyoD was decreased when of MyHC IIA/X-AS lncRNA expression inhibited(P<0.05).Immunofluorescence results show that the fusion and differentiation index of muscle satellite cells to myotubes was significantly reduced.(P<0.05).3.MiR-130 b was predicted to be sponged by MyHC IIA/X-AS lncRNA,and dual luciferase reporter vector systems validate this result;Overexpression of the miR-130 b by agomir in muscle satellite cells,the expression of myogenic differentiation marker genes MyHC and MyoG was significantly decreased(P<0.05).The results of immunofluorescence showed that the number of myotubes was significantly lower compared that in the control group,and the proportion of multinucleated myotubes in the total number of myotubes was consistent(P<0.05).The results showed that miR-130 b inhibited the differentiation of myocytes.4.The target genes of microRNA-130 b were predicted by several bioinformatic software,and 584 target genes were counted.Among which target gene MyHC IIx,and then combined with dual luciferase reporter experiment to confirm miR-130 b interacts with MyHC IIx.The results shown that the expression of MyHC IIA/X-AS and MyHC IIx in LD muscle was significantly higher than that in SM muscle,miR-130 b had opposite trend;then SPSS was used to analyze the expression correlation of MyHC IIA/X-AS/miR-130b/MyHC IIx in different muscle fiber types.The showed that the expression of MyHC IIA/X-AS and MyHC IIx were positively correlated,and both were negative with miR-130 b.After inhibition of MyHC IIA/X-AS lncRNA and overexpression of miR-130 b in muscle satellite cells significantly inhibited the expression of fast muscle-related genes and promoted the expression of slow muscle-related genes(P<0.05).In summary,MyHC IIA/X-AS lncRNA was mainly detected in the cytoplasm of porcine skeletal muscle,which was highly stable and related to host gene expression;MyHC IIA/X-AS lncRNA regulated muscle satellite cells myogenic differentiation through miR-130b;MyHC IIA/X-AS/miR-130b/MyHC IIx was involved in the regulation of different muscle fiber types transformation.The results provided a basis for the exploration of muscle development-related lncRNA and provided a theoretical basis for pork meat quality traits in breeding.