| Skeletal muscle,as the largest motor organ of mammals,is currently the focus of research.Skeletal muscle development is a complex and strictly regulated process that involves the proliferation and differentiation of myoblasts,and fuse into multinucleated muscle fibers with contractile properties.For humans,muscle development disorders usually cause muscle disease that present at or before birth,such as congenital muscular dystrophies and myasthenic syndromes.For livestock,the development of muscle directly impacts the meat yield and commercial value.Perhaps now more than ever,detailed explorations are necessary to guide the disease diagnosis and livestock production.Muscle development is a complex process involving multiple genes and non-coding RNAs(ncRNAs),but the specific mechanisms remain to be explored.Biotechnology and next-generation sequencing have opened a new era in molecular diagnosis of muscle diseases and development gene discovery.For instance,insulin-like growth factor-1 receptor(IGF1R)has been reported to be effective in inducing myoblasts proliferation and differentiation via the phosphoinositide 3’-kinase(PI3K)/AKT pathway.The discovery of new functional genes is not only to improve the diagnostics of these malignant muscle diseases,but also to identify new molecular pathways that may serve as potential therapeutic targets.The PI3K/AKT signaling pathway is an important pathway involved in regulating skeletal muscle development,but there are few studies of non-coding RNAs related to this pathway.Recent reports have suggested that some novel non-coding RNAs,such as micro RNAs(miRNAs)and circular RNAs(circRNAs),are involved in various physiological and pathological processes.Based on sequencing data and bioinformatics analysis,we discovered novel circRILPL1 and circINSR expressed in bovine muscle tissue.However,the functions of the two circRNAs are unclear.The localization of circRNAs in bovine myoblasts was investigated by fluorescence in situ hybridization.Molecular and biochemical assays were used to confirm the role of circRNAs in myoblast proliferation,differentiation and the cell cycle.Mitochondrial membrane potential and annexinⅤ-PE/7-AAD staining assays were performed to assess cell apoptosis.Additionally,interactions between circRNAs,miRNAs,and target mRNAs were examined using bioinformatics,luciferase assay,and RNA immunoprecipitation.Our main results are as follows:1.miR-145 regulates myoblast proliferation and apoptosis via targeting IGF1R.Using the results of high-throughput sequencing of bovine skeletal muscle,miR-145,which is highly expressed in bovine muscle tissue,was screened as the research object.Combining bioinformatics analysis and dual luciferase reporter gene system detection,IGF1R gene was identified as the target gene of miR-145.Moreover,overexpression of miR-145 could significantly reduce the expression of genes related to the PI3K/AKT signaling pathway,such as IRS1,PI3K and AKT.After miR-145 was overexpressed in bovine primary muscle cells,the effects of miR-145 on cell proliferation and apoptosis were examined by real-time qPCR,EdU,CCK-8,JC-1 staining,and flow cytometry.The results indicated that miR-145 could significantly inhibit cell proliferation and cell cycles,and promote cell apoptosis.2.CircRILPL1 promotes muscle proliferation and differentiation via binding miR-145 to activate IGF1R/PI3K/AKT pathway.Based on the theory of ceRNA,if the sequence of circRNAs exactly complementary to the miRNAs seed region,there may be an adsorption relationship.Therefore,we have selected 43 circRNAs that exactly complementary to the miR-145 seed region from the high-throughput sequencing data of bovine muscle tissue in the laboratory.Among them,we identified a novel circRILPL1 which could sponge miR-145 and derived from exon 3 to 4 of RILPL1 gene.Combined with dual luciferase reporter system and RNA-RIP test proved that circRILPL1 could sponge miR-145.Using CCK-8,EdU,RT-qPCR,flow cytometry and immunofluorescence technology,it was found that circRILPL1 could up-regulate the expression of IGF1R gene and activate the PI3K/AKT signaling pathway.Additionally,circRILPL1 was positively correlated with muscle proliferation and differentiation in vitro and could inhibit cell apoptosis.The newly identified circRILPL1 functions as a miR-145 sponge to regulate IGF1R gene,and rescued the inhibitory effect of miR-145 on the PI3K/AKT signaling pathway,thereby promoting myoblasts growth.3.CircINSR promotes muscle proliferation and reduces apoptosis by sponging miR-15/16 to activate IGF1R.In order to further study the non-coding RNAs involved in regulating the IGF1R gene,we focused on circINSR derived from the second exon of the insulin receptor gene(INSR).The INSR gene and IGF1R belong to the insulin-like growth factor family.It is speculated that circINSR may be able to regulate IGF1R gene.Combined with RT-qPCR,EdU,CCK-8and flow cytometry,it was found that circINSR can regulate the expression of IGF1R gene,thereby significantly promoting cell proliferation and inhibiting cell apoptosis.Combined with bioinformatics analysis,it is predicted that the miR-15/16 family may be an intermediate bridge for circINSR to affect IGF1R expression.The dual luciferase reporter system and RNA binding protein co-immunoprecipitation test also finally validated this adsorption relationship.The miR-15/16 family could inhibit muscle cell proliferation and promote muscle cell apoptosis.In short,circINSR alleviates the inhibitory effect of miR-15/16 family on IGF1R gene through targeted adsorption of miR-15/16 family,promotes bovine muscle cell proliferation and inhibits apoptosis.4.Circ INSR promotes proliferation and reduces apoptosis of embryonic myoblasts by sponging miR-34 a.Our data suggested that the expression of miR-34 a decreased after overexpression of circINSR.We speculate that circINSR can also regulate muscle development by targeting miR-34 a.The results show that there are two adsorption sites for circINSR and miR-34 a.Circ INSR and miR-34 a were correspondingly highly expressed in Ago2 immunoprecipitates.miR-34 a has two target genes Bcl-2 and Cyclin E2 related to proliferation and apoptosis.Overexpression of miR-34 a in bovine primary muscle cells can significantly inhibit the expression of apoptosis-suppressing gene Bcl-2,and significantly inhibit the expression of cell proliferation marker genes(Cyclin D1,PCNA and Cyclin E2).The results of EdU and CCK-8 showed that overexpression of miR-34 a significantly inhibited cell proliferation.however,co-transfection of circINSR and miR-34 a increased cell proliferation activity.Overexpression of miR-34 a in muscle cells can cause cell cycle progression to be blocked,and co-transfection of circINSR eliminates some of the inhibitory effects of miR-34 a.Apoptosis test results show that co-transfection of circINSR and miR-34 a can alleviate apoptosis caused by miR-34 a.circINSR regulates cells proliferation and apoptosis through miR-34a-modulated Bcl-2 and Cyclin E2 expression.In this study,based on high-throughput sequencing data,two new circular RNAs were discovered around the key coding genes INSR and IGF1R that regulate muscle development,revealing the role of non-coding RNAs in regulating Qinchuan cattle muscle development.Combining gene transcription and post-transcriptional regulation,ncRNA network and other multi-level regulatory relationships during muscle development,a circRNAs-miRNAs-m RNA-protein regulatory factor multiple regulatory networks were constructed,and the genetic mechanism of Qinchuan beef excellent meat traits was analyzed in depth.Lay a solid theoretical and technical foundation for the promotion of molecular breeding of beef cattle. |
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