Construction Of The CeRNA Network Of Bovine CircRNA And The Functional Mechanism Of Regulating The Development Of Bovine Skeletal Muscle Cells

Most of the skeletal muscle of vertebrates is derived from the paraaxial mesoderm somites,which have undergone the process of hyperplasia and hypertrophy.The number of skeletal muscle cells before birth has been roughly constant through the proliferation and differentiation pathways.After birth,it is mainly the hypertrophy and thickening of muscle fibers and muscle satellites re-entering the cell cycle to repair damaged muscle fibers in response to stimuli.Skeletal muscle development is an extremely complex process that is precisely regulated by transcription factors including myogenic regulatory factor(MRF)and myocyte enhancer factor 2(MEF2)families.Studies have shown that non-codingRNA is also widely involved in the development of skeletal muscle,in which,microRNA(miRNA)and long non-codingRNA(lncRNA)related research is more in-depth.As a new type of circular closed non-codingRNA,circularRNA(circRNA)has attracted great attention due to its high stability characteristics in recent years.The specific molecular mechanism of non-codingRNA regulating skeletal muscle development is currently poorly understood,involving transcription,post-transcriptional and translational regulation.In which,the most widely studied is competitive endogenousRNA(ceRNA),that is,non-codingRNA competitively binds to miRNA through its own miRNA response element(MRE)with other miRNA target molecules,thereby indirectly regulating miRNA target molecules.This paper is based on ceRNA theory and analysis of circRNA sequencing data of Qinchuan cattle skeletal muscle at two developmental periods(90 days for fetal cattle and 24 months old for adult cattle)to construct a circRNA-miRNA-mRNA co-expression network to explore the key circRNA.The main research contents are as follows:1.Construction and functional enrichment analysis of bovine skeletal muscle circRNA-miRNA-mRNA networkIn order to screen the circRNA that functions through the ceRNA pathway,we used bioinformatics to analyze the two developmental stages of Qinchuan cattle(fetal cattle 90 days,adult cattle 24 months old)skeletal muscle circRNA high-throughput sequencing data,and successfully constructed a circRNA-miRNA-mRNA ternary interaction network.Subsequently,GO and KEGG analysis of the network were performed to annotate the potential functions and pathway,and identification and tissue expression profile analysis of these coexpressed circRNAs were performed.The results show that the ternary interaction network is composed of 17 circRNAs,18 miRNAs and 462 mRNAs.Functional enrichment analysis shows that these circRNAs are mainly involved in cell development,suggesting that it may play an important role in the development of bovine skeletal muscle.A total of 6 circRNAs have been identified through experimental methods including circRNA47,circRNA126,circRNA962,circRNA2121,circRNA4394 and circRNA4490.Analysis of the tissue expression profile of Qinchuan cattle at two developmental stages revealed that these circRNAs are highly expressed in fetal bovine skeletal muscle tissue,suggesting that they may regulate early skeletal muscle development.2.Study of the ceRNA mechanism of circHUWE1 regulating the proliferation and differentiation of bovine muscle primary cellsCirc HUWE1-mi R-29b-AKT3 signal axis was screened from the ternary interaction network.circHUWE1(CircRNA47)was identified as circRNA by experimental methods.The nuclear-cytoplasmic separation experiment revealed that circHUWE1 was mainly located in the cytoplasm,suggesting its involvement in post-transcriptional regulation.Bovine muscle cells were transfected with circHUWE1 overexpression vector and inhibitor respectively for functional acquisition and deletion.Cell proliferation,apoptosis and differentiation phenotypes were detected by CCK-8,Edu,Flow cytometry,q RT-PCR and WB.The results showed that circHUWE1 promoted proliferation and inhibited apoptosis and differentiation of bovine muscle cells.Nextly,we used bioinformatics,double luciferase reporter gene assay and AGO2RNA immunoprecipitation(RIP)to verify the interaction between circHUWE1,mi R-29 b and AKT3,the results showed that AKT3 could be released by competitive binding of circHUWE1 with mi R-29 b,and AKT signal pathway could be activated to regulate the proliferation,apoptosis and differentiation of bovine primary muscle cells.3.circSVIL regulates the development of bovine muscle primary cells by inhibiting STAT1 phosphorylation.We screened circSVIL(circRNA126)from the bovine skeletal muscle circRNA-miRNAmRNA co-expression network and successfully carried out biological identification.The results of cytoplasmic separation and FISH showed that circSVIL is expressed in the nucleus and cytoplasm.With the help of CCK-8,Ed U,flow cytometry,q RT-PCR and WB experimental methods,we confirmed that circSVIL can promote the proliferation of primary bovine muscle cells and inhibit cell apoptosis.However,follow-up mechanism exploration shows that circSVIL didn’t play regulatory function through the ceRNA pathway.Bioinformatics predicted that circSVIL has the binding site of STAT1 transcription factor.Through further nuclear and cytoplasmic separation,RIP and rescue experiments,it was confirmed that circSVIL can inhibit STAT1 phosphorylation by interacting with STAT1,inhibiting STAT1 nuclear translocation and affect the downstream signal cascade of STAT1,thereby promoting the proliferation of muscle primary cells and inhibiting apoptosis.In our study,the circRNA-miRNA-mRNA ternary network model was constructed by using the circRNA high-throughput sequencing data of skeletal muscle circRNA in the two developmental stages of Qinchuan cattle,and the circHUWE1 and circSVIL that have the potential to regulate skeletal muscle development were screened out.A series of functional mechanism studies have been carried out at the muscle primary cell level to clarify the function and mechanism of circHUWE1 and circSVIL in regulating the development of bovine muscle primary cells.These findings provide a new regulatory pathway for bovine skeletal muscle development,and provide potential candidate molecules for subsequent beef cattle breeding.