College of Animal Science and Technology, Northwest A&F University, Yangling, 712100, China.
Department of Livestock Management, Breeding and Genetics, The University of Agriculture, Peshawar, 25130, Pakistan.
Int J Mol Sci. 2019 Sep 4;20(18):4338. doi: 10.3390/ijms20184338.
The TORC2 gene is a member of the transducer of the regulated cyclic adenosine monophosphate (cAMP) response element binding protein gene family, which plays a key role in metabolism and adipogenesis. In the present study, we confirmed the role of in bovine preadipocyte proliferation through cell cycle staining flow cytometry, cell counting assay, 5-ethynyl-2'-deoxyuridine staining (EdU), and mRNA and protein expression analysis of proliferation-related marker genes. In addition, Oil red O staining analysis, immunofluorescence of adiponectin, mRNA and protein level expression of lipid related marker genes confirmed the role of in the regulation of bovine adipocyte differentiation. Furthermore, the transcription start site and sub-cellular localization of the gene was identified in bovine adipocytes. To investigate the underlying regulatory mechanism of the bovine , we cloned a 1990 bp of the 5' untranslated region (5'UTR) promoter region into a luciferase reporter vector and seven vector fragments were constructed through serial deletion of the 5'UTR flanking region. The core promoter region of the gene was identified at location -314 to -69 bp upstream of the transcription start site. Based on the results of the transcriptional activities of the promoter vector fragments, luciferase activities of mutated fragments and siRNAs interference, four transcription factors ( and ) were identified as the transcriptional regulators of gene These findings were further confirmed through Electrophoretic Mobility Shift Assay (EMSA) within nuclear extracts of bovine adipocytes. Furthermore, we also identified that and regulate gene as activators in the promoter region. We can conclude that gene is potentially a positive regulator of adipogenesis. These findings will not only provide an insight for the improvement of intramuscular fat in cattle, but will enhance our understanding regarding therapeutic intervention of metabolic syndrome and obesity in public health as well.
TORC2 基因是调节环磷酸腺苷反应元件结合蛋白基因家族的转导子的成员，在代谢和脂肪生成中发挥关键作用。在本研究中，我们通过细胞周期染色流式细胞术、细胞计数测定、5-乙炔基-2'-脱氧尿苷染色（EdU）以及增殖相关标记基因的 mRNA 和蛋白表达分析，证实了在牛前体脂肪细胞增殖中的作用。此外，油红 O 染色分析、脂联素免疫荧光、脂质相关标记基因的 mRNA 和蛋白水平表达证实了在调节牛脂肪细胞分化中的作用。此外，我们在牛脂肪细胞中鉴定了的转录起始位点和亚细胞定位。为了研究牛的潜在调节机制，我们将 1990bp 的 5'非翻译区（5'UTR）启动子区域克隆到荧光素酶报告载体中，并通过串联缺失 5'UTR 侧翼区域构建了七个载体片段。鉴定出位于转录起始位点上游-314 至-69bp 的基因核心启动子区域。基于启动子载体片段的转录活性、突变片段和 siRNAs 干扰的荧光素酶活性的结果，鉴定出四个转录因子（ 和 ）是基因的转录调节因子。这些发现通过牛脂肪细胞核提取物中的电泳迁移率变动分析（EMSA）进一步得到证实。此外，我们还发现和调节作为启动子区域的基因的激活剂。我们可以得出结论，基因可能是脂肪生成的正调控因子。这些发现不仅为提高牛的肌内脂肪提供了深入的了解，而且还增强了我们对代谢综合征和肥胖症在公共健康中的治疗干预的理解。
