Molecular Mechanism Of MiR-483 Regulating The Proliferation And Differentiation Of Goat Primary Myoblasts

Lamb is an important part of livestock and poultry meat,and its quality and yield are directly related to the growth and development of skeletal muscle.microRNA(miRNA)is a kind of short single-stranded non-coding RNA,which regulates gene expression at the post-transcriptional level by binding to the 3’UTR of target genes.miR-483 is a conserved and highly expressed microRNA in mammals.Existing studies have shown that miR-483 plays an important role in the growth and development of mammalian skeletal muscle,but the study of miR-483 on the growth and development of goat skeletal muscle has not been reported.In this study,goat primary myoblasts were used as materials.The molecular and cellular biological methods such as chemical synthesis mimics,inhibitor,dual luciferase reporter and gene editing were used to systematically explore the role and molecular regulatory mechanism of miR-483 in the proliferation and differentiation of goat myoblasts.The main results are as follows:1.miR-483 negatively regulates the proliferation and differentiation of goat primary myoblastsThe mimics of miR-483 were transfected into goat primary myoblasts to over-express miR-483,or the inhibitor of miR-483 was transfected into goat primary myoblasts to inhibit the expression of miR-483.Compared with the relevant control group,over-expression of miR-483 reduced the proliferation and differentiation of goat primary myoblasts.Inhibition of miR-483 expression resulted in enhanced proliferation and differentiation of goat primary myoblasts,indicating that miR-483 negatively regulates proliferation and differentiation of goat primary myoblasts.2.miR-483 targeted IGF1 gene and down-regulated IGF1 expression.Bioinformatics software was used to predict IGF1 as the target gene of miR-483,and IGF1 wild type and mutant luciferase reporter vectors were constructed to identify the targeting relationship between miR-483 and IGF1 gene.The results showed that overexpression of miR-483 could significantly inhibit the luciferase activity of wild type,but the fluorescence activity of mutant type had no change.IGF1 was the target gene of miR-483.q RT-PCR and Western-blot were used to detect the regulatory effect of miR-483 on target gene IGF1.Overexpression of miR-483 decreased the m RNA and protein levels of IGF1,while inhibition of miR-483 increased the m RNA and protein levels of IGF1.These results indicated that miR-483 down-regulated the expression of IGF1 gene.3.IGF1 knockout inhibited the proliferation and differentiation of goat myoblasts.IGF1-KO goat primary myoblasts were obtained by CRISPR/Cas9 gene editing technology.Compared with normal goat primary myoblasts,the proliferation and differentiation of IGF1-KO goat primary myoblasts were weakened,indicating that IGF1 gene knockout inhibited the proliferation and differentiation of goat primary myoblasts.4.miR-483 inhibited the expression of related proteins in IGF1/PI3K/Akt signaling pathway.Overexpression and inhibition of miR-483 were used to detect the protein changes of PI3 K and p-Akt in the subsequent pathways.In normal goat primary myoblasts,overexpression of miR-483 led to significant down-regulation of p-Akt and PI3 K expression,and inhibition of miR-483 led to significant up-regulation of p-Akt and PI3 K expression.However,inhibition of miR-483 expression in IGF1-KO goat primary myoblasts did not increase the expression of p-Akt and PI3 K,indicating that miR-483 inhibited the expression of related proteins in IGF1/PI3K/Akt signaling pathway.In summary,this study showed that miR-483 negatively regulates the proliferation and differentiation of goat myoblasts.The specific molecular mechanism may be that miR-483 targets IGF1 gene and regulates the expression of key proteins in IGF1/PI3K/Akt signaling pathway,thereby affecting the proliferation and differentiation of goat primary myoblasts.This study enriches the theoretical knowledge of molecular regulation of muscle development and provides a reference for promoting goat breeding and growth regulation.