Screening Of Key CircRNAs In Skeletal Muscle Fiber Types Of Pigs And Functional Study Of CircMYLK4

Muscle tissues mainly consist of three types: skeletal muscle,cardiac muscle and smooth muscle.Skeletal muscle accounts for about 40%-60% of the body weight.Skeletal muscle is made up of large number of different types of muscle fibers.There are four types of skeletal muscle fibers in pig: slow oxidation type,rapid oxidation type,rapid glycolysis type and intermediate type.Among them,the proportion of slow oxidized muscle fibers is high,which helps to improve muscle tenderness and juiciness.Therefore,the composition of the muscle fiber type plays a decisive role in meat quality.CircRNA is a newly discovered RNA molecule that does not contain a 5' end cap structure and a 3' end poly(A)tail and is joined by covalent bonds to form a closed loop structure.It acts as a molecular sponge of miRNA and RNA binding proteins and is involved in the regulation of gene expression.It is highly conserved,specific and stable.It plays an important regulatory role at both transcriptional and post-transcriptional levels.In this study,a high-throughput sequencing technique was used to construct a cDNA library of two different skeletal muscles,the longissimus dorsi(LD)and the soleus muscle(Sol).After quality control filtration of the sequencing data,the differential circRNA was screened and identified.The interaction with the miRNA network,the target gene GO function and KEGG pathway analysis were performed based on the differential circRNA.This would reveal the regulatory mechanism affecting different skeletal muscle fiber types in pigs,and provide an experimental basis for improving pork quality.It was found that the expression of circMYLK4 in Sol was significantly higher than that in LD through sequencing analysis(P<0.05).In order to verify the effect of circMYLK4 on different skeletal muscle fiber types in pigs,we first inject circMYLK4-AAV into piglets to explore its effects on different types of muscle fibers.Secondly,we detect the expression profile of circMYLK4 in different tissues of pigs,the localization in satellite cells and its expression pattern of muscle satellite cells during differentiation;afterwards,the effect of circMYLK4 on the differentiation of porcine muscle satellite cells was explored by using overexpression techniques.The above studies have the following main results:1.A total of 86645908 and 72841678 fragments are generated from the cDNA library in the longissimus dorsi(LD)and soleus(Sol),respectively,among them 26,015 and 25,130 circRNAs were identified by alignment analysis.A total of 10,388 circRNAs were co-expressed in two muscles.Further analysis of the screened circRNA revealed 181 differentially expressed circRNAs between LD and Sol,of which 90 were up-regulated and 91 were down-regulated.2.Pathway enrichment analysis showed that the differentially expressed circRNAs are potentially involved in the regulation of porcine skeletal muscle fiber types through signaling pathways such as AMPK,FoxO,and PI3K-Akt.3.Sequencing data and RT-qPCR results showed that circMYLK4 was significantly higher in Sol than in LD(P<0.05);circMYLK4 was widely expressed in different tissues of pigs,with the highest in slow as well as fast muscle tissues in pigs.circMYLK4 is highly expressed in porcine satellite cells during late stages of differentiation and is mainly localized in the cytoplasm.4.In vivo injection of circMYLK4-AAV showed that circMYLK4 significantly increased the expression of the slow muscle related genes MyHCI,Tnnt1,Tnni1,Tnnc1,Myl3(P<0.05)and mitochondria-related genes PGC-1?,Tfam,Atp5 o,Cycs,Ndufa5(P<0.05),indicating that circMYLK4 can contribute to the development of slow-twitch muscle fibers.5.Overexpression of circMYLK4 significantly increased the expression of the slow muscle marker gene MyHCI(P<0.05),on the other hand significantly decreased the expression of the differentiation marker genes MyoD and MyoG(P<0.05),indicating that overexpression of circMYLK4 can inhibit the differentiation of porcine skeletal muscle satellite cells.In summary,this study screened the key circRNAs that regulate pig muscle fiber types through high-throughput sequencing;circMYLK4 promotes the development of slow muscle fibers in pigs;circMYLK4 inhibits the differentiation of porcine skeletal muscle satellite cells.