The Mechanism Of HuR In Promoting The Differentiation Of Skeletal Muscle Satellite Cells In Goat

The growth and development of muscle is an extremely complex and precise process,which is affected by a regulatory network composed of a variety of regulatory factors,including growth factors,transcription factors,regulatory proteins,and some hormone receptors.In recent years,a large number of coding genes and non-codingRNAs(miRNA,lncRNA,and circRNA)have been found to participate in the regulation network of muscle production.As one of theRNA-binding proteins,Hu antigen R(HuR)has been widely studied and plays an important regulatory role in muscle development and a variety of biological processes.at present,studies have shown that HuR regulated muscle growth and development,but there are few reports on its research in the muscle development of domestic animals,especially in goats.This experiment uses goat skeletal muscle satellite cells(MuSCs)as the experimental material.The effects of HuR on the differentiation of MuSCs were revealed by qPCR,MyHC immunofluorescence,Western Blot,RNA sequencing(RNA-seq),RNA immunoprotein co-precipitation andRNA stability test,to analyze the molecular mechanism of HuR regulating the differentiation of goat MuSCs.The experimental results are as follows:(1)The expression profile of HuR in different tissues(longissimus dorsi,psoas,semimembranosus,semitendinosus,gastrocnemius,brain,liver,lung,and kidney)of goat.Significantly,HuR expression was higher in muscle than in other tissues.In addition,we examined the expression of HuR in the longissimus dorsi muscle(LD)of different developing goat tissues(E90,E105,E135,B3,B150(E for embryonic stage and B for postnatal stage),the expression level of HuR peaked on day 90 of gestation.Furthermore,the expression levels of HuR during goat MuSC differentiation were measured,and the results indicated that the expression of HuR fluctuates during MuSC differentiation;(2)Overexpression of HuR significantly upregulated the mRNA levels of myogenic marker genes MyoG,MyoD,and MyHC during MuSCs differentiation(P <0.05).Knockdown of HuR significantly downregulated the expression of myogenic marker genes MyoG,MyoD,and MyHC(P < 0.05).Furthermore,Protein validation and immunofluorescence assay results of MyHC indicated its concordance with mRNA expression levels;(3)RNA-seq was utilized to analyze differentially expressed genes(DEGs)upon HuR knockdown during MuSCs differentiation.The result shows that 144 differential genes,113 downregulated genes and 31 upregulated genes were identified in HuR knockdown cells compared with the control cells,8 differential genes related to muscle development were identified,and 3 differential genes(Myomaker,CHRNA1,CAPN6)were significantly downregulated(P < 0.01).Further online tool prediction and RIPqPCR assay confirmed that Myomaker bound to HuR(4)The mRNA and protein expression of Myomaker were significantly increased in overexpression of HuR during differentiation of MuSCs(P < 0.05).furthermore,the mRNA and protein expression of Myomaker were significantly decreased in interference of HuR during differentiation of MuSCs(P < 0.05).RNA stability assays showed that the stability of Myomaker was significantly decreased when inhibition of HuR compared with the control.In addition,we verified that HuR could enhance myogenic gene mRNA(MyoD,MyoG)stability and further promote goat MuSCs differentiation.(5)The co-transfection test results showed that the mRNA expression of MyoG and MyHC decreased when inhibiting Myomaker.The overexpression of HuR eliminated the inhibitory effect of Myomaker on MuSCs.Therefore,HuR promotes goat MuSCs differentiation by increasing the stability of Myomaker.In conclusion,in the present study,we demonstrate that HuR is a crucial posttranslational regulator of muscle differentiation and identify a novel target of HuR in the promotion of muscle differentiation in goats.Mechanistically,HuR binds to Myomaker and increases the mRNA stability of Myomaker,thereby activating MuSC differentiation by positively regulating the expression of Myomaker.The result provides a reference basis for the molecular mechanism of HuR in regulating the development of goat skeletal muscle,meanwhile,it is of great significance to refine the molecular regulatory network of goat muscle growth and development.