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.

The elongation of very long chain fatty acids protein 6 (ELOVL6) gene encodes a key enzyme that plays a role in lipogenesis through the catalytic elongation of both saturated and monounsaturated fatty acids. Previous studies have described the high expression of bovine ELOVL6 in adipose tissues. However, transcriptional regulation and the functional role of ELOVL6 in lipid metabolism and adipocyte proliferation remain unexplored. Here, a 1.5 kb fragment of the 5′-untranslated region promoter region of ELOVL6 was amplified from the genomic DNA of Qinchuan cattle and sequenced. The core promoter region was identified through unidirectional 5′-end deletion of the promoter plasmid vector. In silico analysis predicted important transcription factors that were then validated through site-directed mutation and small interfering RNA interference with an electrophoretic mobility shift assay. We found that the binding of KLF6 and PU.1 transcription factors occurred in the region −168/+69. Both perform a vital regulatory function in the transcription of bovine ELOVL6. Overexpression of ELOVL6 significantly upregulated the expression of peroxisome proliferator activated receptor γ (PPARγ), but inhibited the expression of fatty acid-binding protein 4 (FABP4), while silencing of ELOVL6 negatively regulated the messenger RNA expression level of PPARγ, FABP4, ACSL, and FATP1. In addition, ELOVL6 promotes adipocyte proliferation by regulating the cell-cycle genes' expression. Taken together, these findings provide useful information about the transcriptional regulation and functional mechanisms of bovine ELOVL6 in lipid metabolism and adipocyte proliferation in Qinchuan cattle. K E Y W O R D S bovine adipocytes, elongation of very long chain fatty acids protein 6, fatty acid metabolism, promoter J Cell Biochem. 2019;120:13932-13943. wileyonlinelibrary.com/journal/jcb 13932 |

In this review, we highlight information on microRNA (miRNA) identification and functional characterization in the beef for muscle and carcass composition traits, with an emphasis on Qinchuan beef cattle, and discuss the current challenges and future directions for the use of miRNA as a biomarker in cattle for breeding programs to improve meat quality and carcass traits. MicroRNAs are endogenous and non-coding RNA that have the function of making post-transcriptional modifications during the process of preadipocyte differentiation in mammals. Many studies claim that diverse miRNAs have an impact on adipogenesis. Furthermore, their target genes are associated with every phase of adipocyte differentiation. It has been confirmed that, during adipogenesis, several miRNAs are differentially expressed, including miR-204, miR-224, and miR-33. The development of mammalian skeletal muscle is sequentially controlled by somite commitment into progenitor cells, followed by their fusion and migration, the proliferation of myoblasts, and final modification into fast- and slow-twitch muscle fibers. It has been reported that miRNA in the bovine MEG3-DIO3 locus has a regulatory function for myoblast differentiation. Likewise, miR-224 has been associated with controlling the differentiation of bovine adipocytes by targeting lipoprotein lipase. Through the posttranscriptional downregulation of KLF6, miR-148a-3p disrupts the proliferation of bovine myoblasts and stimulates apoptosis while the miR-23a~27a~24-2 cluster represses adipogenesis. Additional to influences on muscle and fat, bta-mir-182, bta-mir-183, and bta-mir-338 represent regulators of proteolysis in muscle, which influences meat tenderness.

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