Mechanism Of Circ-CDR1as Regulating Goat Skeletal Muscle Satellite Cells Differentiation

Muscle growth and development as a determinant physiological indicator of the meat quality of livestock animals,its molecular regulation mechanism has been regarded as the basis and breakthrough of animal molecular breeding and improvement.The growth and development of goat skeletal muscle is mediated by a variety of regulatory factors,including growth factors,transcription factors,regulatory proteins and some hormone receptors,and so on.Previous studies on molecular mechanisms have always been focused on DNA,mRNA,and miRNA levels.In recent years,further studies of long non-coding RNAs(lncRNAs)have also begun to emerge.However,studies on the regulation of muscle growth by circular RNAs have rarely been reported.In this study,based on transcriptome sequencing of the different developmental stages of goat skeletal longissimus dorsi,a well-studied circular CDR1 as which hold a high level in brains and harboured more than 70 miR-7 binding sites for regulating the expression of downstream target genes,was screened out due to specific spatial and temporal expression level.Then the function of CDR1 as and correlative regulatory mechanism were studied by conducting on goat skeletal muscle satellite cells(SMSCs).The main results are as follows:(1)The goat CDR1 as undergoes backsplicing and forms a length of 1481 nucleotides circular RNA.The exonuclease digestion experiment revealed that it had strong anti-nuclease exonuclease ability which further confirmed the circular agent.In contrast to humans and mice,the bases near the cyclization sites of the goat CDR1 as sequence are the conserved.(2)The expression trend of CDR1 as in longissimus dorsi from the embryo 45 d to the fetus 90 d day were proved to rise first and then decline,accumulated a high contant in mid-embryonic development(E75~E105)(P<0.01).In the tissue expression profiling of E75,CDR1 as is widely expressed,among which the expression in the longissimus dorsi muscle is extremely significantly higher than in other tissues and even exceeds that in the brain.CDR1 as was expressed at a low level during the proliferative phase of goat SMSCs,but the expression level was significantly upregulated when SMSCs differentiation(P<0.01).(3)After overexpression of CDR1 as,some genes related to myogenic differentiation were up-regulated.MyHC immunofluorescence staining further showed that overexpression of CDR1 as significantly promoted SMSCs differentiation.(4)CDR1as knockdown significantly impaired myoblast fusion myogenic differentiation marker genes,consistently,results were also strengthened by IF staining for MyHC,CDR1 as knockdown significantly impaired myoblast fusion.(5)It has been predicted that there are 70 conserved binding sites for goat CDR1 as and miR-7.Subcellular location study showed that most CDR1 as exist in the cytoplasm.Fluorescence in situ hybridization experiments showed that both CDR1 as and miR-7 are located in the cytoplasm and their positions are highly coincident.(6)Dual-luciferase reporter gene system verified that miR-7 could bind to the3?-UTR of IGF1 R gene,thereby inhibiting the expression of IGF1 R.(7)When MiR-7 mimics transfected SMSCs,myogenic differentiation was inhibited by downregulating IGF1 R expression.Overexpression of CDR1 as can rescue the inhibition of SMSCs differentiation by miR-7 mimics.It is suggested that CDR1 as may promote SMSCs differentiation by competitively binding to miR-7 and impeding the downregulation of IGF1 R.In short,through this experiment,we obtained the goat CDR1 as sequence,predict and verify that CDR1 as can block the downregulation of IGF1 R by competitive binding to miR-7 to promote the differentiation of SMSCs.This study have enriched the functional studies involved in circular RNA and have important implications for further elucidating the molecular mechanism of animal muscle cell differentiation.