miR-92a激活Wnt/β-catenin通路抑制低氧运动脂肪细胞分化与脂肪形成的机制研究

Hypoxic exercise significantly reduces body weight and body fat, improves lipid metabolism compared to normoxic exercise. Wnt/β-catenin pathway is a switch and major negative regulation factor of fat cells differentiation, its mechanism that Wnt/β-catenin pathway regulates fat cells differentiation in the hypoxic training is still unclear. The microarray and real time PCR results showed that there is a significantly decreased expression of miR-92a in the hypoxic exercise more than normal exercise. The result of Double luciferase assay showed that the target gene of miR-92a is Fzd10 which is an acceptor in the Wnt pathway. The mRNA expression of the c-myc which located at Wnt pathway downstream is increased significantly. So we build hypothesis that miR-92a decreased fat cells differentiation and adipogenesis of hypoxic training obese body by activating Wnt/β-catenin signaling pathway. This study aims to detect related gene and protein expression through miR-92a over-expression and lower-expression experiments in 3T3-L1 cell and hypoxic training obesity rats, find the direct target genes of Wnt signaling pathway using ChIP-Seq technology, then analyze the mechanism of adipose cells differentiation and adipogenesis regulated by miR-92a via Wnt/β-catenin signaling pathway, and further perfect the theory system on differentiation and adipogenesis, and establish theoretical basis for weight and fat loss in hypoxic trainining.

低氧运动比常氧运动降低体重体脂、改善脂代谢的作用更为显著。Wnt/β-catenin通路是脂肪细胞分化的开关和主要负调控因子，其在低氧运动中的机制研究比较少。前期，课题组利用微阵列芯片和PCR检测发现高脂饮食大鼠低氧运动比常氧运动miR-92a表达显著性降低，双荧光素酶实验证实miR92a靶向Wnt信号通路受体Fzd10，Wnt通路下游基因c-myc的mRNA表达显著性升高。因此，提出研究假说：miR-92a通过调控Fzd10激活Wnt信号通路抑制低氧运动脂肪细胞分化和脂肪形成。本项目拟利用3T3-L1细胞、低氧运动肥胖大鼠模型，过表达和抑制表达miR-92a，检测相关基因和蛋白表达，并应用染色体免疫共沉淀测序技术（ChIP-Seq）获得Wnt信号通路的靶向基因，从而探讨miR-92a通过Wnt通路调控脂肪细胞分化和脂肪形成的机制，为低氧运动降重减脂健康促进提供理论依据。

目前超重和肥胖持续攀升成为全球性问题，人群肥胖率显著增加。低氧运动能够改善脂代谢，抑制脂肪酸合成，促进脂肪酸分解和氧化，具有明显的降体重、减体脂的作用。本研究利用3T3-L1细胞、低氧运动肥胖大鼠模型，过表达和抑制表达miR-92a，检测相关基因和蛋白表达，探讨miR-92a通过Wnt/β-catenin信号通路调控低氧运动脂肪细胞分化和脂肪形成的分子机制。经双重荧光素酶报告基因检测，Fzd10基因被证实为miRNA-92a的靶基因。低氧环境下miR-92a的mRNA表达水平降低，其靶基因Fzd10表达的上调，引起位于Wnt/β-catenin通路下游的c-Myc的mRNA表达水平显着增加。在3T3-L1脂肪细胞中，过表达和抑制表达miR-92a，结果发现miR-92a过表达会抑制 PPARγ、C/EBPα、FABP4以及 AP2、ADD1/SREBP1、FAS等脂肪细胞分化和脂肪形成相关基因的表达，而抑制miR-92a则会提升或恢复相关基因的表达。基于此我们初步认为，miR-92a可能通过调节Wnt/β-catenin信号传导途径导致脂解，影响脂肪代谢。在此基础上，本研究深入解析miR-92a在低氧运动肥胖大鼠脂肪组织中对脂肪代谢的调控机制，通过构建低氧肥胖运动大鼠模型和分析脂肪组织中蛋白质组学变化，确认Wnt /β-catenin信号通路发生了变化。脂肪组织Real time PCR检测结果显示：低氧运动组比常氧运动组Wnt/β-catenin信号通路相关分子c-myc、β-catenin、Wnt3a的mRNA表达量显著升高，GSK3β、APC、Wnt4、Wnt5a的mRNA表达量显著降低。Western blot检测结果显示：低氧运动组比常氧运动组c-myc、β-catenin、FAS、LPL、SREBP1、PPARγ、FZD10蛋白表达量显著升高，低氧运动组比常氧运动组ATL、CD36、CPT-1蛋白表达量无显著性差异。比较分析低氧运动肥胖大鼠和常氧运动肥胖大鼠全基因组DNA的CpG岛甲基化水平，发现Wnt信号通路相关的基因CpG和DMR甲基化明显增高。本项目表明在低氧运动环境下，Wnt/β-catenin信号通路被激活，miR-92a通过Wnt/β-catenin信号通路在细胞水平和动物水平调控低氧环境的脂肪代谢。本研究为寻找减脂提供新的思路和方法。