Mechanisms Study On Of Regulation Of Myoblast Proliferation,Differentiation And Apoptosis By CircACLY In Jingyuan Chickens

Inosine monophosphate(IMP),as an important purine nucleotide,is not only an important taste substance,but also an important product of purine nucleotide metabolism,and is the basis for the survival and reproduction of life.Studies have shown that the IMP de novo synthesis pathway affects cell proliferation and apoptosis.The IMP de novo synthesis pathway is influenced by multiple gene expression and signaling,and gene expression levels are regulated by multiple factors,with post-transcriptional regulation and epigenetic modifications are important mechanisms affecting the level of gene expression.Non-coding RNAs(ncRNAs)including microRNA(miRNA),circular RNA(circRNA)and long-stranded non-coding RNA(lncRNA)have been found to be involved in the post-transcriptional regulation of genes,and an increasing number of studies have shown that these ncRNAs are involved in the growth and development of skeletal muscle.Jingyuan chicken is a national livestock and poultry protection breed and one of the five livestock and poultry protection breeds in Ningxia region.In this study,RNA-seq technology and bioinformatics analysis were used to screen key circRNAs affecting the proliferation,differentiation and apoptosis of skeletal muscle cells in Jingyuan chicken,as test materials.Meanwhile,cellular function validation of key circRNAs and miRNAs was also performed using overexpression assays,knockdown assays,quantitative Real-time PCR(RT-PCR),dual-luciferase gene reporter system,RNase R digestion assays,subcellular localization assays,CCK-8 assays,EDU assays,Elisa assays,etc.The main findings are as follows.(1)In this study,transcriptome sequencing(RNA-seq)technology was used to identify the expression profile of circRNAs in the muscle tissue of Jingyuan chicken,and a total of 9456 circRNAs were identified,of which 2111 circRNAs were identified in the breast muscle,2148 circRNAs were identified in the leg muscle,and 5197 circRNAs were identified in both the breast and leg muscles.A total of 516 differentially expressed circRNAs were screened,of which 188 were up-regulated and 328 were down-regulated.The circRNA-miRNA-mRNA interaction network analysis yielded a total of 42 differentially expressed circRNAs,6 differentially expressed miRNAs and 12 differentially expressed genes,among which neurogranin(NRGN),pyruvate kinase(PKM2)and ectonucleoside triphosphate diphosphohydrolase 7(ENTPD7)were the most differentially expressed.In addition,RT-PCR results showed consistent sequencing and quantification of circRNAs,which can be used for subsequent functional validation of circRNAs.(2)PCR amplification by divergent primers and convergent primers and RNase R digestion assays identified circACLY as circRNA from exons 2-3 of the ATP citrate lyase(ACLY)gene.Subcellular localization assays on circACLY showed that circACLY is mainly found in the cytoplasm.In addition,wild-type/mutant dual-luciferase reporter gene vectors for circACLY and ENTPD7 were successfully constructed,and the dual-luciferase reporter gene system assay confirmed that gga-miR-6660-3p could directly bind to the binding sites of circACLY and ENTPD7,and circACLY could adsorb gga-miR-6660-3p to enhance the expression level of ENTPD7.(3)The RT-PCR,CCK-8,EDU,Elisa and induction of differentiation assays revealed that gga-miR-6660-3p was differentially expressed in different tissue sites and at different developmental stages in Jingyuan chickens,and that gga-miR-6660-3p inhibited the IMP de novo synthesis pathway to promote myoblast apoptosis and suppressed myoblast proliferation and differentiation.(4)The RT-PCR,CCK-8,EDU and Elisa assays revealed that circACLY was differentially expressed in different tissue sites and at different developmental stages in Jingyuan chickens.circACLY promoted myoblast proliferation,differentiation and IMP de novo synthesis pathway and eliminated the proliferation,differentiation and IMP ab initio synthesis pathway of myoblasts by gga-miR-6660-3p inhibitory effects.In addition,circACLY inhibited myocyte apoptosis and eliminated the apoptosis-promoting effect of gga-miR-6660-3p on myocytes.In conclusion,this study systematically investigated that circACLY sponge adsorbed gga-miR-6660-3p regulated IMP de novo synthesis pathway in Jingyuan chicken to affect the proliferation,differentiation and apoptosis of myoblasts,enriched the regulatory mechanism of circRNA in skeletal muscle development and IMP synthesis in local chicken species,and provided some theoretical basis for the genetic improvement of livestock and poultry meat quality.