Research Of CircDYRK1A As A MiR21-5p Molecular Sponge Regulates KLF5 To Affect Proliferation And Differentiation Of Bovine Myoblast

The improvement in quality of life has increased the demand for beef,and meat production performance,as one of the important economic traits of meat livestock,has been the focus of animal breeding research.Meanwhile,muscle yield and quality have become important factors affecting the economic efficiency of Chinese livestock industry.However,the regulatory network associated with muscle development is extremely complex,and in addition to gene initiation and expression changes,there are many related regulatory factors that modify site-specific processes,including DNA methylation and histone modifications.Translated with www.Deep L.com/Translator(free version)Circular RNAs(circRNAs)are a special class of non-coding RNA molecules that widely expressed in eukaryotes and involved in regulating a large number of physiological and pathological activities of the organism,however,researches on the regulation of muscle development in beef cattle by circRNA were poorly reported.By analyzing the transcriptome sequencing of skeletal muscle of fetal cattle(90 days of embryonic period)and adult cattle period(24 months of age)completed in the laboratory previous study,the differentially expressed circRNA set was obtained by comparison,and after further screening,this study found that the expression of circRNAs formed by double specificity tyrosine phosphorylation regulated kinase 1A(DYRK1A)exons 11 and 12 was significantly decreased in adult bovine skeletal muscle.Then,by overexpressing or interfering with circDYR1 KA,the molecular biology techniques such as RT-q PCR and Western blot were used to determine its regulatory role on the proliferation and differentiation of bovine myoblast.To further determine the mechanism related to circDYR1 KA regulation of bovine adult myoblast development,molecular experimental methods such as dual luciferase reporter gene technique were used to reveal that circDYR1 KA as a miR21-5p molecular sponge,adsorb it and regulate KLF5 gene expression.Finally,the effect of circDYR1 KA on muscle regeneration process in mice was explored by injecting circDYR1 KA overexpression plasmid into mice.The main findings were as follows.1.Bovine circDYRK1 A promotes myoblast differentiation and inhibits proliferationIn this study,circDYRK1 A was screened for subsequent study based on the existing circRNA sequencing results in the laboratory.After sequence comparison,circDYRK1 A was found to be loop-forming from exons 11 and 12 of the DYRK1 A gene.Then,the loop-forming site of circDYRK1 A was identified using Sanger sequencing,and circDYRK1 A was amplified using circDYRK1 A divergent primers in RNase R digestion treatment and cell nucleoplasm isolation samples to further verify the authenticity of circDYRK1 A.Further,after overexpression or interference of circDYRK1 A in bovine myoblast,molecular experimental techniques such as RT-q PCR,Western blot,and CCK-8 were used to confirm that circDYRK1 A could promote differentiation and inhibit proliferation of bovine myoblast.2.circDYRK1 A binding to miR21-5p affects proliferation and differentiation of bovine myoblast.Combining the results of nucleoplasmic separation amplification assay and fluorescence in situ hybridization(FISH),it was hypothesized that circDYRK1 A acts as a miRNA molecular sponge in the cytoplasm,performs miRNA functional regulation.Based on the circRNA and miRNA sequence data,the RNAhybrid website was used to predict miRNAs with potential binding relationship with circDYRK1 A,and miR134 and miR21-5p were found to be the potential binding miRNAs of circDYRK1 A.miR21-5p was identified by dual luciferase reporter gene technique to circDYRK1 A by excellent binding properties.Then,molecular experimental techniques such as RT-q PCR,Western blot,and CCK-8 were used to confirm that circDYRK1 A regulates bovine myoblast proliferation and differentiation through adsorption of miR21-5p.3.circDYRK1A-miR21-5p-KLF5 ternary regulatory chain affects proliferation and differentiation of bovine myoblastAfter determining the adsorption of miR21-5p by circDYRK1 A,the downstream target genes of miR21-5p were predicted by Target Scan website,and the targeting relationship between KLF5 and miR21-5p was further determined by using dual luciferase reporter gene technique and RNA immunoprecipitation.Then,molecular experimental techniques such as RT-q PCR,Western blot,and CCK-8 were used to determine that circDYRK1 A could affect the proliferation and differentiation of bovine myoblast by regulating KLF5 gene expression through adsorption of miR21-5p.4.In vivo experiments verify that circDYRK1 A promotes muscle cell regenerationThe effect of circDYRK1 A on muscle cell regeneration was further investigated using in vivo experiments.First,sequence similarity between bovine-derived and murine-derived circDYRK1 A was identified,the sequence similarity was 90%.C57BL/6 mice were used as experimental subjects,and a muscle injury model was established by intramuscular injection of cardiotoxin CTX.Then,pc DNA2.1-circDYRK1A/null was injected in the tibialis anterior muscle,and RT-q PCR and HE staining were used to confirm that circDYRK1 A promotes myoblast differentiation,which in turn helps muscle regeneration.In summary,circDYRK1 A,a miR21-5p molecular sponge,regulates KLF5 gene expression by adsorbing miR21-5p,thereby promoting differentiation and inhibiting proliferation of bovine myoblast.This study shows that circDYRK1 A is a regulator of muscle development,and elucidates the mechanism of action of the circDYRK1A-miR21-5p-KLF5 regulatory axis,providing new basic theoretical material for uncovering the complex regulatory network and molecular mechanisms regulating bovine muscle development.