miR-460b-5p promotes proliferation and differentiation of chicken myoblasts and targets RBM19 gene

The meat production of broilers is crucial to economic benefits of broiler industries, while the slaughter performance of broilers is directly determined by skeletal muscle development. Hence, the broiler breed-ing for growth traits shows a great importance. As a kind of small noncoding RNA, microRNA (miRNA) can reg-ulate the expression of multiple genes and perform a wide range of regulation in organisms. Currently, more and more studies have confirmed that miRNAs are closely associated with skeletal muscle development of chickens. Based on our previous miR-seq analysis (accession num-ber: PRJNA668199), miR-460b-5p was screened as one of the key miRNAs probably involved in the growth regula-tion of chickens. However, the regulatory effect of miR-460b-5p on the development of chicken skeletal muscles is still unclear. Therefore, miR-460b-5p was further used for functional validation at the cellular level in this study. The expression pattern of miR-460b-5p was investigated in proliferation and differentiation stages of chicken pri-mary myoblasts. It was showed that the expression level of miR-460b-5p gradually decreased from the proliferation stage (GM 50%) to the lowest at 24 h of differentiation. As differentiation proceeded, miR-460b-5p expression increased significantly, reaching the highest and stabiliz-ing at 72 h and 96 h of differentiation. Through mRNA quantitative analysis of proliferation marker genes, CCK-8 and Edu assays, miR-460b-5p was found to significantly facilitate the transition of myoblasts from G1 to S phase and promote chicken myoblast proliferation. mRNA and protein quantitative analysis of differentiation marker genes, as well as the indirect immunofluorescence results of myotubes, revealed that miR-460b-5p significantly stimulated myotube development and promote chicken myoblast differentiation. In addition, the target relation-ship was validated for miR-460b-5p according to the dual-luciferase reporter assay and mRNA quantitative analysis, which indicates that miR-460b-5p was able to regulate RBM19 expression by specifically binding to the 30 UTR of RBM19. In summary, miR-460b-5p has posi-tive regulatory effects on the proliferation and differentia-tion of chicken myoblasts, and RBM19 is a target gene of miR-460b-5p.