Study On The Mechanism Of Bovine CircSGCB Regulating Muscle Cell Proliferation And Differentiation Through KLF3

The growth and development of skeletal muscle are regulated by many factors.For the livestock and poultry industry,it reflects the quality and yield of meat produced by animals.As a new key candidate factor,circRNAs could regulate the development of muscle cells.However,although many circRNAs have been detected in bovine muscle tissues,the research on the regulatory function and mechanism of circRNA on muscle cells is still in the preliminary stage.In this study,by analyzing the RNA-seq transcript expression data of circRNAs in fetal and adult skeletal muscles of Qinchuan cattle measured in the previous laboratory researches,a differentially expressed circRNA(circSGCB)was screened out that is related to muscle development.The authenticity of circSGCB was identified through full-length amplification of circSGCB,RNase R tolerance test,and expression detection.Using loss of function experiment that interfere with circSGCB,as assessed by q PCR,Western-blot,Ed U,CCK-8,flow cytometry,MYHC immunofluorescence,and other techniques to explore the effects of circSGCB on the proliferation and differentiation of cattle muscle cells.In addition,through bioinformatics prediction,expression detection,and dual luciferase report analysis,the targeting relationship of circSGCB-miR-27a-3p-KLF3 was detected.Furthermore,through over-expression and interference technology,the effects of bta-miR-27a-3p and the target molecule KLF3 on the proliferation and differentiation of cattle muscle cells were studied.Finally,the use of bioinformatics analysis,preliminary tests(q PCR,Western-blot detection),and rescue tests to study the role of circSGCB-miR-27a-3p-KLF3 in the PI3K/AKT pathway.These studies might indicate that bovine circSGCB regulates KLF3 through the ce RNA mechanism and affects the development of bovine myoblasts.These main investigate findings are as follows:1.circSGCB regulated cell proliferation and differentiation by adsorbing bta-miR-27a-3p in bovine muscle cellsResearch verified the authenticity of circSGCB.The circSGCB is derived from the second exon of its maternal gene SGCB,with a length of 210 nt.Using loss of function experiment that interfere with circSGCB,it was indicated that bovine circSGCB could indeed inhibit the proliferation of Qinchuan cattle myoblasts and promote muscle differentiation.Furthermore,the nuclear and cytoplasmic localization test showed that the circSGCB was existed in the cytoplasm of bovine muscle cells.Therefore,the sequence of circSGCB was analyzed by RNAhybrid software and found that there was a binding site for miR-27a-3p.Relying on dual-luciferase report and AGO2 RIP detection,it was found that circSGCB and bta-miR-27a-3p had a binding effect.2.bta-miR-27a-3p could regulate the proliferation and differentiation of bovine muscle cells through targeted inhibition of KLF3Using the test for the gain of function that over-expressed with bta-miR-27a-3p,the study found that bta-miR-27a-3p could promote the proliferation of bovine muscle cells and inhibit its differentiation.Furthermore,through Targetscan software prediction,it was found that bta-miR-27a-3p could target the 3’ UTR of KLF3 gene.Then,it was found that the two did indeed have a binding effect by the dual fluorescence detection test.Moreover,by over-expression of bta-miR-27a-3p,it was found that the expression level of KLF3 was significantly declined.3.KLF3 inhibited the proliferation of bovine muscle cells and promoted their differentiation,and circSGCB-miR-27a-3p-KLF3 participated in the PI3K/AKT pathwayThe use of overexpression and interference with the KLF3 gene in bovine muscle primary cells,it was showed that KLF3 might inhibit the proliferation of cattle primary myoblasts and promote cell differentiation.Through the mouse CHIP-seq database and bioinformatic analysis,it was found that KLF3 could bind to the promoter region of PIK3R2 gene.After over-expression and interference with KLF3,it was found that KLF3 might inhibit the transcription of PIK3R2 gene and inhibit PI3K/AKT pathway-related proteins(IRS1/PDK1/PI3K/AKT)expression.Then,after interference with circSGCB or over-expression of miR-27a-3p,PI3 K and AKT protein were also found to be affected.Furthermore,through co-transformation experiments in bovine muscle cells,it was found that over-expression of KLF3 could rescue the up-regulation of proliferation-related genes caused by interference with circSGCB,and attenuate the signal transduction of PI3K/AKT pathway.Therefore,circSGCB might release miR-27a-3p’s inhibition of KLF3 expression by binding miR-27a-3p,thereby affecting the PI3K/AKT pathway to regulate the proliferation and differentiation of cattle muscle cells.Further,these findings improved the complex molecular network of cattle muscle development to provide a theoretical basis for the development of the Chinese beef cattle industry and molecular breeding in the future.