MicroRNA bta-miR-365-3p inhibits proliferation but promotes differentiation of primary bovine myoblasts by targeting the activin A receptor type I

Abstract

Background: MicroRNAs act as post-transcriptional regulators that repress translation or degrades mRNA transcripts. Each microRNA has many mRNA targets and each mRNA may be targeted by several microRNAs. Skeletal muscles express a plethora of microRNA genes that regulate muscle development and function by controlling the expression of protein-coding target genes. To expand our understanding of the role of microRNA, specifically bta-miR-365-3p, in muscle biology, we studied its function in regulating primary bovine myoblast proliferation and differentiation.Results: We first show that bta-miR-365-3p is predominantly expressed in skeletal muscle and heart tissue in Chinese Qinchuan beef cattle. Quantitative PCR and western blotting showed that overexpression of bta-miR-365-3p significantly reduced the levels of cyclinD1 (CCND1), cyclin dependent kinase 2 (CDK2) and proliferating cell nuclear antigen (PCNA) but stimulated the expression of muscle differentiation markers MYOD1, MYOG, both at mRNA and protein level. Moreover, downregulation of bta-miR-365-3p increased the expression of CCND1, CDK2 and PCNA but decreased the expression of MYOD1 and MYOG at both mRNA and protein levels. Furthermore, flow cytometry, EdU proliferation assays and immunostaining showed that increased levels of bta-miR-365-3p suppressed cell proliferation but promoted myotube formation, whereas a decreased level of bta-miR-365-3p had opposite consequences. Finally, we determined that activin A receptor type I (ACVR1) is a direct target of bta-miR-365-3p. Thus, dual luciferase gene reporter assays demonstrated that bta-miR-365-3p can bind to the 3'UTR of ACVR1 gene to regulate its expression. Consistently, knock-down of ACVR1 was associated with reduced CDK2, CCND1 and PCNA expression but increased MYOG and MYOD1 expression both at mRNA and protein level.Conclusion: Collectively these data suggest that bta-miR-365-3p represses proliferation but promotes differentiation of bovine myoblasts through a mechanism involving downregulation of ACVR1.

Goat AKAP12: Indel Mutation Detection, Association Analysis With Litter Size and Alternative Splicing Variant Expression

“…The 179/192-bp DNA sequence containing the 13-bp region was cloned into psiCHECK-2 using T4 DNA Ligase and the insert was released by XhoI and NotI digestion (TaKaRa Biotech Co., Ltd.). The miR-181 sequence was cloned into the pcDNA3.1 vector (Invitrogen) as described previously (Kang et al, 2020;Hao et al, 2021), and the insert was released by KpnI and XhoI digestion.…”