Chi-miR-99b-3p Regulates the Proliferation of Goat Skeletal Muscle Satellite Cells In Vitro by Targeting Caspase-3 and NCOR1.

Abstract

Simple SummaryIn this study, we investigated the role of chi-miR-99b-3p in goat skeletal muscle satellite cells (SMSCs). We confirmed that chi-miR-99b-3p promoted proliferation through targeting Caspase-3 and nuclear receptor corepressor 1, and inhibiting the intrinsic apoptosis-related genes in SMSCs, whereas inhibition of chi-miR-99b-3p had the opposite effect. Furthermore, integrative transcriptomic analysis revealed that overexpression of chi-miR-99b-3p induced various differentially expressed (DE)-gene-associated processes. In addition, 47 dysregulated miRNAs, including 16 upregulated and 31 downregulated miRNAs, which were involved in the biological pathways associated with the DE genes, were identified. Our study demonstrated that chi-miR-99b-3p was an effective facilitator of goat SMSCs. This study is helpful for future studies in skeletal muscle development.We previously found that chi-miR-99b-3p was highly expressed in the skeletal muscle of 7-month-old (rapid growth period) goats and speculated that it may be associated with muscle development. To further investigate the role of chi-miR-99b-3p in goats, we found that chi-miR-99b-3p acted as a myogenic miRNA in the regulation of skeletal muscle development. Dual-luciferase reporter assays, qRT-PCR, and Western blot results confirmed that Caspase-3 and nuclear receptor corepressor 1 were direct targets for chi-miR-99b-3p as their expression was inhibited by this miR. Cell proliferation and qRT-PCR assays showed that chi-miR-99b-3p promoted proliferation through relevant targets and intrinsic apoptosis-related genes in goat skeletal muscle satellite cells (SMSCs), whereas inhibition of chi-miR-99b-3p had the opposite effect. Furthermore, integrative transcriptomic analysis revealed that overexpression of chi-miR-99b-3p induced various differentially expressed (DE) genes mainly associated with the cell cycle, relaxin signaling pathway, DNA replication, and protein digestion and absorption. Notably, most of the cell-cycle-related genes were downregulated in SMSCs after miR-99b-3p upregulation, including the pro-apoptosis-related gene BCL2. In addition, 47 DE miRNAs (16 upregulated and 31 downregulated) were determined by Small RNA-sequencing in SMSCs after chi-miR-99b-3p overexpression. Based on the KEGG enrichment analysis, we found that these DE miRNAs were involved in the biological pathways associated with the DE genes. Our study demonstrated that chi-miR-99b-3p was an effective facilitator of goat SMSCs and provided new insights into the mechanisms by which miRNAs regulate skeletal muscle growth in goats.