The Effect And Mechanism Of Lnc-ORA On Skeletal Muscle Fiber Type Transformation And Glycolipid Metabolism

Skeletal muscle is a highly heterogeneous tissue,accounting for about 40% of body weight,composed of muscle fibers,sarcolemma,muscle satellite cells and nerves.As a heterogeneous tissue,skeletal muscle is made up of two types of fibers,type I and type II muscle fibers.These two types of fiber have different glucolipid metabolism pathways.The former is rich in mitochondria and mainly relies on aerobic metabolism for energy,while the latter mainly relies on anaerobic glycolysis for energy.At the same time,the type and composition ratio of muscle fibers also affect the quality of meat products,including sensory scores and nutritional scores.Long noncoding RNAs(lnc RNAs)are defined as RNAs ranging in length from 200 nt to 100 kb that are transcribed by mechanisms similar to m RNAs but lack significant protein-coding potential.Studies have found that lnc RNAs function in a variety of biological processes,including regulation of cell growth,proliferation,differentiation and apoptosis,tissue and organ development.However,the lnc RNAs that regulate the transformation of animal skeletal muscle fiber types still need to be identified,and the related mechanisms need to be further studied.Preliminary research in the laboratory found that after adeno-associated virus(AAV)-mediated lnc-ORA was overexpressed in mice,the muscle tissue of mice showed differential changes in color and size.At the same time,it was found that the expression of lnc-ORA in fast-twitch muscle fibers was significantly higher than that in slow-twitch muscle fibers,suggests that lnc-ORA may have a potential function in regulating the transformation of muscle fiber types.Therefore,this study took myoblasts,porcine muscle satellite cells and lnc-ORA knockout mice constructed by the CRISPR/Cas9 system as the research objects.Through the research methods of overexpression,interference and gene knockout.The metabolic cage test,mouse endurance test,Insulin and glucose tolerance test,fluorescent double-labeling,HE staining,mitochondrial and enzyme activity detection and other technical methods were used to explore the role and molecular mechanism of lnc-ORA in regulating the composition of skeletal muscle fiber types at the cellular,molecular and in vivo levels.The main results obtained are as follows:1.Knockout of lnc-ORA promotes muscle growth.A lnc-ORA knockout mouse(lncORA-KO)was successfully constructed using the CRISPR/Cas9 system.Compared with wild-type mice(WT),lnc-ORA-KO mice had reduced body weight and significantly decreased adipose tissue weight.However,the weight of muscle tissue such as gastrocnemius and extensor digitorum longus increased significantly,and the cross-sectional area of the gastrocnemius muscle fibers increased significantly.2.Knockout of lnc-ORA promotes the conversion of fast-twitch to slow-twitch muscle fibers.The expression of lnc-ORA was significantly higher in fast muscle(extensor digitorum longus)than in slow muscle(soleus).Compared with WT mice,the proportion of slow-twitch muscle fibers in the gastrocnemius muscle of KO mice was significantly increased,and the proportion of fast-twitch muscle fibers was decreased.The cross-sectional area of the soleus muscle of KO mice was larger than that of WT mice.The expression of slow muscle related genes in GAS of mice in KO group was significantly higher than that in WT group.At the same time,the activity of succinate dehydrogenase(SDH)in skeletal muscle of lnc-ORA-KO mice was significantly higher than that of WT group,and the activity of lactate dehydrogenase(LDH)was lower than that of WT group.The expression of mitochondrial respiratory chain complex-related proteins CIII-UQCRC,CII-SDHB and CI-NDUFB8 in skeletal muscle increased after lnc-ORA knockout.The related genes Ndufa1,Ndufs4,Ndufaf4,Sdhd,Cox7a1 and Atp5e encoding mitochondrial respiratory chain complexes were up-regulated.Besides,lnc-ORA-KO mice exhibited stronger exercise tolerance compared with WT mice.3.The oxidative metabolism of lnc-ORA knockout mice is enhanced.Glucose tolerance test(GTT)showed that compared with WT mice,KO mice were more able to metabolize glucose,and there were significant differences in serum cholesterol and lactate dehydrogenase between KO mice and WT mice.At the same time,the expression of glucose transport-related gene Glu1 in the skeletal muscle of mice after knocking out lnc-ORA increased,and the expression of fatty acid oxidation,uptake and utilization genes Cpt1 a,Cpt1b,and Acads were significantly up-regulated.Metabolic cage test showed that lnc-ORA-KO mice had higher oxygen consumption and calorific value and lower respiratory entropy than WT mice,and thermal imaging showed that the body surface temperature of lnc-ORA-KO mice was significantly higher than that of WT mice.4.lnc-ORA may promote myoblast muscle fiber type transformation through interaction with PKM.Lnc-ORA increases fast-twitch myotubes production and inhibits slow-twitch myotubes production in myoblasts.At the same time,the expression level of fast-twitch muscle differentiation-related marker protein Fast-My HC increased,and the expression levels of fast-twitch muscle contraction-related genes Tnni2 and Tnnt3 increased.The expression level of slow muscle differentiation-related marker protein Slow-My HC decreased,and the expression of slow muscle contraction related genes Tnni1 and Tnnt1 were down-regulated.The expression of the gene PGC-1α,which regulates the transformation of fast-twitch muscle fibers to slow-twitch muscle fibers,was significantly decreased.Mechanistically,Lnc-ORA can adsorb protein PKM to promote the transformation of fast and slow myotubes in myoblasts.5.Exogenous overexpression of lnc-ORA promotes fast-twitch muscle fiber differentiation in porcine muscle satellite cells.Immunofluorescence results showed that exogenous overexpression of lnc-ORA promoted the generation of fast-twitch muscle fibers in porcine muscle satellite cells.At the same time,the expression level of fast-twitch muscle fiber differentiation-related protein Fast-My HC increased,and the expression levels of fasttwitch muscle contraction-related genes Tnni2 and Tnnt3 increased significantly.In conclusion,this study demonstrated that the functional loss of lnc-ORA can promote the transformation of fast to slow muscle fibers in skeletal muscle and the mitochondrial biogenesis of skeletal muscle,and promote skeletal muscle glucose and lipid metabolism and systemic energy metabolism in mice.It was also found that lnc-ORA overexpression promoted the formation of fast muscle myoducts in C2C12 myoblasts and myosatellite cells.The results of this study provide a new candidate target for improving pork quality.