Whole Transcriptome Analysis Of Chicken Embryonic Skeletal Muscle And Study On The CeRNA Mechanism Of Key LncRNA TRSMD Regulating Myoblast Proliferation And Differentiation

Chicken provides humans with a large amount of high-quality animal protein every year,and it also plays an important role in China’s meat consumption.There are many local chicken breeds in China,which generally have the advantages of good meat quality and strong resistance.However,the growth rate of local chickens is relatively slow,so they lack competitiveness in the market.With the increasing demand for high-quality chicken,improving the production performance of Chinese local chickens has become one of the main problems faced by the broiler industry.The growth and development of skeletal muscle directly affects chicken meat production,and it is crucial for the broiler industry.Revealing the molecular mechanism of skeletal muscle growth and development has important scientific significance and guidance for the breeding of Chinese local chickens.Studies have found that non-coding RNA is ubiquitous in organisms,widely participating in many regulatory processes,including the skeletal muscle development.In this experiment,the leg muscles of Bian chicken at 14-day（S14vsF14）and 20-day（S20vsF20）embryonic ages were collected from the fast-and slow-growing groups for whole transcriptome sequencing.We constructed ceRNA regulatory networks of differentially expressed lncRNA,miRNA,and mRNA between fast-and slow-growing groups.A key regulatory axe lncRNA TRSMD-miR24-3p-BRT-1 was finally screened,and multiple molecular biology experiments（RT-qPCR,Flow Cytometry,Edu,CCK-8,Indirect Immunofluorescence and Western Blot）were conducted to reveal their mechanism and function in chicken primary myoblasts（CPMs）.The main results are as follows:1.Transcriptome sequencing analysis of lncRNA and mRNA in leg muscles of Bian chicken during embryonic stage.This study analyzed the differentially expressed（DE）lncRNA and mRNA in leg muscles of fast-growing and slow-growing Bian chicken at 14-day and 20-day embryonic ages.And 64 and 50 DE lncRNAs were screened in S14vsF14 and S20vsF20,among which,4 co-differentially expressed lncRNAs were found.The differential expression of mRNA in S14vsF14 and S20vsF20 showed that 268 and 256 DE mRNAs were screened,including 34 co-differentially expressed mRNAs.GO and KEGG pathway enrichment analysis obtained some terms related to skeletal muscle development,including biological processes（BP）terms cell development and regulation of multicellular organismal process,and pathway terms Wnt signaling pathway and signaling pathway and Calcium signaling pathway.Co-location target analysis of DE IncRNAs and DE mRNA was performed,and 4 and 9 lncRNA-mRNA pairs were obtained in groups S14vsF14 and S20vsF20,respectively,including TCONS₀0195013-SPOPL,TCONS₀0080797-SEMA5 A and TCONS₀0030135-LIMS1.2.Transcriptome sequencing analysis of miRNA for leg muscles of Bian chicken during embryonic stageIn this study,the DE miRNAs in leg muscles of S14vsF14 and S20vsF20 was identified and the enrichment analysis of target genes was also performed.A total of 61 and 55 DE miRNAs were identified in the two comparison groups S14vsF14 and S20vsF20.Among them,10 co-differentially expressed miRNAs were found.The GO function enrichment analysis for the targets of DE miRNAs revealed some BP entries related to skeletal muscle development;Analysis of KEGG pathway enrichment showed that there are 8 common pathways in TOP20 between S14vsF14 and S20vsF20 groups,including Hedgehog signaling pathway,Cell adhesion molecules（CAMs）and Tryptophan metabolism.Combining the DE mRNAs,we constructed miRNA-mRNA networks by predicting DE miRNAs and DE mRNAs target relationships.The important pairs gga-miR-12265-5pXLOC₁0820 and gga-miR-12265-5p-XLOC₀43993 in S14vsF14 were screened.In S20vsF20 group,we found novel₁16-XLOC₀01679 and gga-miR-24-3p-SBF1 pairs.3.Screening of key ceRNA regulatory axes during myoblast development and validation of their targeting relationshipsThis study constructed ceRNA regulatory networks with DE lncRNAs,miRNAs,and mRNAs,and validated the targeting relationship of the key regulatory axis lncRNA TRSMD-miR-24-3p-BRT-1.RT-qPCR was used to detect the expression of miR-24-3p,lncRNA TRSMD,and BRT-1 in tissues of 1-day chick.And results showed that the expression levels of them in skeletal muscle（leg muscle and breast muscle）were all high.The changes of miR-24-3p,lncRNA TRSMD,and BRT-1 during the proliferation and differentiation of CPMs were also detected and the results found that the trend of lncRNA TRSMD and BRT-1 increased first and then decreased in CPMs,while miR-24-3p has the opposite trend.Therefore,it is speculated that they may have a target relationship and are also related to the development of myoblasts.The nucleocytoplasmic separation experiment and FISH showed that lncRNA TRSMD mainly exists in the cytoplasm.After transfection of miR-24-3p mimic in CPMs,it was found that both lncRNA TRSMD and BRT-1 were significantly reduced,while after transfection of miR-24-3p inhibitor,the expression of lncRNA TRSMD and BRT-1 was significantly increased.Then the dual luciferase experiment and RIP experiment successfully verified the targeted binding sites between miR-24-3p and lncRNA TRSMD/BRT-1.Finally,the rescue experiments of lncRNA TRSMD and BRT-1 were conducted by co-transfecting mimic+pcDNA3.1-lncRNA or mimic+pcDNA3.1-BRT-1,successfully validating the targeted regulatory relationship among them.4.Function of lncRNA TRSMD-miR-24-3p-BRT-1 axis in chicken primary myoblastsIn this study,functions of lncRNA TRSMD,miR-24-3p and BRT-1 were explored with molecular experiments after transfecting the interfering and overexpression vectors in CPMs.Results showed that both lncRNA TRSMD and BRT-1 significantly increased the expression of proliferation marker genes CCNA2,CCNB2,and CDK1,and inhibited the expression of P21 during myoblast proliferation.And miR-24-3p downregulated the expression of CCNA2,CCNB2,and CDK1,while upregulated the expression of P21.Subsequently,flow cytometry,EdU and CCK-8 experiments showed that both lncRNA TRSMD and BRT-1 could promote the proliferation of CPMs,while miR-24-3p inhibited the proliferation of CPMs.During the differentiation of CPMs,it was found that lncRNA TRSMD and BRT-1 significantly inhibited the expression of MYOD,MYOG and MYHC,while miR-24-3p increased their expression.At the same time,western blot results showed that MYHC protein expression was consistent with the trend of mRNA level.Subsequently,indirect immunofluorescence revealed that lncRNA TRSMD and BRT-1 could reduce myotube area of CPMs,while miR-24-3p increased it.In summary,lncRNA TRSMD and BRT-1 can inhibit the differentiation of CPMs,while miR-24-3p promotes its differentiation.