Bta‐miR‐149‐5p inhibits proliferation and differentiation of bovine adipocytes through targeting CRTCs at both transcriptional and posttranscriptional levels

Abstract

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.

MiR-146a-5p targeting SMAD4 and TRAF6 inhibits adipogenensis through TGF-β and AKT/mTORC1 signal pathways in porcine intramuscular preadipocytes

“…miRNAs function generally by binding target genes that match their "seed sequences" to suppress or degrade target gene mRNAs after transcription and coordinate normal processes, including cellular proliferation, differentiation and apoptosis [7,8]. Increasing evidence has shown that miRNAs, including miR-206 [9], miR-149-5p [10] and miR-204-5p [11], play key roles in preadipocyte proliferation and differentiation. Moreover, many miRNAs are differentially expressed in porcine adipose tissue during different developmental stages and have been studied using transcriptome sequencing technology [12].…”

Bovine Pre-adipocyte Adipogenesis Is Regulated by bta-miR-150 Through mTOR Signaling

“…The treatment method of CCK-8 cell cycle detection was the same as that of EdU. The specific operation steps were carried out according to the reference (Khan et al, 2020). The kit used was TransDetect Cell Counting Kit (Transgen biotechnology, Beijing, China), and the detection instrument was an Infinite R 200PRO (Tecan Trading AG, Switzerland).…”

“…Advancements to high-throughput sequencing technology has allowed deeper exploration of cellular regulatory processes (Katz et al, 2010). Previous studies have already revealed the regulatory effects of human miRNA, including miR-204 , miR-27a (Kim et al, 2010), miR-210 (Ren et al, 2020), miR-24 (Liu et al, 2020), miR-378 (Duarte et al, 2020), miR-149-5p (Khan et al, 2020), miR-143 (Zhang L. et al, 2020), and miR-145 (Wang et al, 2020), on adipogenesis. These classic mechanisms also occur in the adipose tissue, where they have a large impact on the surrounding tissue (Arcidiacono et al, 2020;Raza et al, 2020).…”

Differentially Expressed miRNA-Gene Targets Related to Intramuscular Fat in Musculus Longissimus Dorsi of Charolais × Holstein F2-Crossbred Bulls

“…This study confirms the expression pattern of various miRNAs that have already been implicated in adipogenesis and fat tissue. For example, increased levels of let-7a in IMF and subcutaneous fat tissue [ 25 , 40 ], miR-101 in serum and adipose tissue of obese individuals [ 41 ], miR-1282 in nonalcoholic fat deposition in liver [ 42 ], miR-149 in rumen and fat tissue compared to liver, spleen, lungs, kidney and small intestine tissues [ 43 ], miR-504 in adipose-derived mesenchymal stem cells of obese individuals [ 44 ], miR-132 in the omental fat of obese individuals [ 45 ] and miR-421 in fatty hepatic tissues of high fat diet (HFD)-diet mice [ 46 ] have been reported. Moreover, miR-10b [ 47 ], miR-30c [ 48 , 49 ] miR-132 [ 50 ], miR-149 [ 51 ], miR-331 [ 52 ], miR-335 [ 53 ] and miR-425 [ 54 ] were all upregulated in adipogenesis and accelerated the process of adipogenic differentiation.…”