MiR-487b-3p Inhibits Proliferation And Differentiation Of Goat Primary Myogenic Cells By Targeting The IRS1

Skeletal muscle is the most abundant tissue in mammals and plays a key role in the metabolism.Investigating the factors regulating skeletal muscle development in goats and the related molecular mechanisms is of great significance to promote lamb growth and development,high-quality goat breeding and even livestock development.microRNAs(miRNAs)are important non-coding RNAs that are endogenously expressed and play an important role in the regulation of post-transcriptional gene expression.Currently,a large number of miRNAs have been identified as important factors in the regulation of myogenic cell proliferation and differentiation.miR-487b-3p is widely present in muscle tissue and plays a regulatory role in many biological processes such as cell proliferation and differentiation.However,its function and molecular mechanisms in goat myogenesis are not yet clear.Investigating the specific function and regulatory network of miR-487b-3p in goat skeletal muscle can help to understand the regulatory process of goat myogenesis and provide a reference for the molecular breeding of high-quality goats.In this study,the potential role of miR-487b-3p in the proliferation and differentiation of primary myogenic cells was investigated by performing gain-of-function and loss-of-function experiments targeting miR-487b-3p and analyzing miR-487b-3p expression differences in goat primary myogenic cells using quantitative reverse transcription PCR,Western-Blot,CCK-8,EdU,and cell cycle analysis.Afterwards,miR-487b-3p targets were identified by bioinformatics and dual-luciferase assays,and its function in proliferation and differentiation of primary myogenic cells was examined using siRNA-mediated knockdown strategies.The specific molecular mechanisms of miR-487b-3p involvement in the proliferation and differentiation of goat primary myogenic cells were further explored by using CRISPR/Cas9 gene editing technology with DsRed-Puror-eGFP(RPG)reporter system to construct miR-487b-3p-KO cell model,and finally the following conclusions were obtained:(1)miR-487b-3p inhibits myogenic cell proliferation and differentiation:miR-487b-3p was enriched in skeletal muscle,and the expression level of miR-487b-3p was much higher in lambs than in adult goats;overexpression of miR-487b-3p inhibited myogenic cell division and myotube formation,down-regulated the expression of PCNA,CDK2 and other proliferation-related genes,up-regulated p27 In contrast,inhibition of miR-487b-3p expression significantly promoted the division and myotube formation of goat myogenic cells.(2)Targeting relationship between miR-487b-3p and IRS1: Dual luciferase reporter assays showed that miR-487b-3p and IRS1 have a reciprocal relationship.Overexpression of miR-487b-3p significantly down-regulated IRS1 expression levels;in contrast,inhibition of miR-487b-3p significantly up-regulated IRS1 expression levels.(3)Knockdown of IRS1 inhibited the proliferation and differentiation of myogenic cells: IRS1 was concentrated in muscle tissue;knockdown of IRS1 decreased cell proliferation,down-regulated the expression of proliferation-related genes such as PCNA and CDK2,and up-regulated the expression of p27 gene;meanwhile,the expression of differentiation-related genes MyoG and MyHC was down-regulated.In addition,the IRS1/PI3K/Akt pathway was significantly inhibited after knockdown of IRS1.(4)miR-487b-3p is a negative regulator that inhibits the growth of goat adult myoblasts and regulates the proliferation and differentiation of goat adult myoblasts by inhibiting the IRS1/PI3K/Akt signaling pathway:miR-487b-3p-KO significantly promoted cell proliferation and the expression of cell cycle-related genes PCNA,CDK2,PCNA and Cyclin E,and significantly activation of IRS1/PI3K/Akt pathway expression.In conclusion,this study systematically investigated the function and regulatory mechanism of miR-487b-3p in the proliferation and differentiation of primary myogenic cells in goats,providing a reference and reference for skeletal muscle growth and development studies and molecular breeding in goats.