RESUMEN
The proliferation and differentiation of myoblasts are essential for the regeneration and development of skeletal muscles. However, the process of skeletal muscle development in cattle is complex and needs to be further investigated. The microRNAs (miRNAs) are endogenous, small noncoding RNAs that play a critical role during skeletal muscle development. In this study, we evaluated the function of miR-885 in muscle development in cattle. The results found that the expression of miR-885 was gradually upregulated during myoblast proliferation, whereas progressively downregulated during myoblast differentiation. The overexpression of miR-885 promoted cell proliferation of myoblast in cattle. Moreover, we further noted that the overexpression miR-885 triggered the expression level of various marker genes involved in cell proliferation, including proliferating cell nuclear antigen (PCNA), cyclin-dependent kinase 2 (CDK2), and cyclin B1 (CCNB1). Furthermore, it was observed that overexpression of miR-885 inhibited cell differentiation, and significantly decreased messenger RNA and protein expression levels of myogenic differentiation 1 (MyoD1) and myogenin (MyoG) in primary bovine myoblasts. Moreover, the miR-885 inhibitor revealed that miR-885 inhibited cell proliferation and promoted cell differentiation. In addition, the overexpression of miR-885 markedly decreased MyoD1 expression in primary bovine myoblasts. The luciferase reporter assay, quantitative real-time polymerase chain reaction, and western blot (WB) further indicated that miR-885 directly binding to 3' UTR of MyoD1 gene during transcriptional regulation. Conclusively, these results signified that miR-885 could be critical for the proliferation and differentiation in primary bovine myoblast cells by targeting the MyoD1 gene in cattle.
Asunto(s)
Diferenciación Celular/genética , Proliferación Celular/genética , MicroARNs/genética , Proteína MioD/genética , Mioblastos/patología , Regiones no Traducidas 3'/genética , Animales , Bovinos , Regulación hacia Abajo/genética , Regulación de la Expresión Génica/genética , Desarrollo de Músculos/genética , Miogenina/genética , ARN Mensajero/genética , Transcripción Genética/genética , Regulación hacia Arriba/genéticaRESUMEN
MicroRNAs are small, single stranded, and noncoding RNAs that have been proven to be potent regulators of adipogenesis. However, the role of bta-miR-149-5p in regulating bovine adipogenesis is still unclear. Expression profiling in different stages of adipogenesis revealed that bta-miR-149-5p was enriched in the proliferation stage, and also on Day 9 of differentiation in bovine adipocytes. Our gain of function study showed that bta-miR-149-5p can negatively regulate both bovine adipocyte proliferation and differentiation. Overexpression of bta-miR-149-5p suppressed the expression of proliferation marker genes at both the messenger RNA (mRNA) and protein levels, markedly decreased the percentage of S-phase cells, decreased the number of EdU-stained cells, and substantially reduced adipocyte proliferation vitality in the cell count assay. Collectively, these findings elucidated that bta-miR-149-5p inhibits adipocyte proliferation. Furthermore, overexpression of bta-miR-149-5p also suppressed the expression of adipogenic genes at both the mRNA and protein levels, inhibited lipid accumulation, and reduced the secretion of adiponectin in bovine adipocytes. Furthermore, a luciferase activity assay explored how bta-miR-149-5p targeted CRTCs (CRTC1 and CRTC2) directly. This targeting was further validated by the mRNA and protein level expression of CRTC1 and CRTC2, which were down regulated by bta-miR-149-5p overexpression. Moreover, bta-miR-149-5p indirectly targeted CRTC1 and CRTC2 through regulating their key transcription factors. Overexpression of bta-miR-149-5p suppressed the expression of SMAD3, while enriched the expression of NRF1, which are the key transcription factors and proven regulators of CRTC1. Overexpression of bta-miR-149-5p also repressed the expression of C/EBPγ, XBP1, INSM1, and ZNF263, which are the key regulators of CRTCs, at both the mRNA and protein levels. These findings suggest that bta-miR-149-5p is a negative regulator of CRTC1 and CRTC2 both at transcriptional and posttranscriptional level. Taken together, these findings suggest that bta-miR-149-5p can regulate adipogenesis, which implies that bta-miR-149-5p could be a target for increasing intramuscular fat in beef cattle.