MicroRNA-24通过Wnt4/β-catenin信号通路促进棕色脂肪分化及分子机制研究

Obesity, associated with various diseases, is a chronic metabolic disorder. Brown adipocyte differentiation plays a protective role in obesity. We found that the expression of miR-24 increased significantly in the process of brown adipocyte differentiation induced by BMP7, while the transcription activity of proliferational genes were inhibited in our previous study. Wnt/β-catenin signaling pathway has been reported involving in the process of adipogenesis by down-regulating the activation of proliferational genes. While recently it has been testified that miR-24 can attenuates neointimal hyperplasia in diabetic rat by inhibiting Wnt4/β-catenin signaling pathway. Thus, we hypothesize that miR-24 may promote brown adipocyte differentiation by inhibiting Wnt4/β-catenin signaling pathway. To test our hypothesis, firstly the role of miR-24 in brown adipogenesis and its effect on Wnt4/β-catenin signaling pathway will be elucidated through transfecting miR-24-mimic and miR-24-inhibitor into brown preadipocytes. Secondly, the interaction between Wnt4 and miR-24 will be analyzed by RNA-ChIP and Luciferase reporter genes. Finally, we conducted the miR-24 knockout mice with C57BL6 background ,recording the body weight and metabolism after intervention with high fat diet, to verify miR-24 can promote brown adipocyte differentiation and prevent obesity by regulating Wnt4/β-catenin signaling pathway.

棕色脂肪分化已成为肥胖症治疗的新靶点。我们在前期研究中发现，在BMP7诱导棕色脂肪分化的过程中，miR-24表达显著上调，而增殖基因表达被明显抑制；研究发现Wnt/β-catenin信号激活可能通过促进增殖基因表达而影响脂肪分化；新近报道miR-24通过抑制Wnt4/β-catenin减轻糖尿病大鼠血管内膜增生。因此我们提出miR-24可能通过抑制Wnt4/β-catenin信号通路，从而促进棕色脂肪分化。本课题拟将棕色脂肪前体细胞miR-24过表达和沉默后探讨miR-24对棕色脂肪分化及Wnt4/β-catenin信号通路的影响；进而采用RNA-ChIP和荧光素酶报告基因等手段验证miR-24与Wnt4的直接作用关系；最后观察高脂喂养的miR-24基因敲除C57BL6小鼠的体重和糖脂代谢情况，进一步证实miR-24通过Wnt4/β-catenin影响棕色脂肪分化，进而影响肥胖及糖脂代谢。

前期研究发现棕色脂肪分化中mir24表达显著增加且呈时间依赖性；并发现脂肪前体细胞中细胞增殖期的mir24表达增加更显著，而增殖基因表达被明显抑制机制不明确。我们提出mir24可能通过抑制Wnt4通路调节棕色脂肪分化的假设。.为验证mir24对Wnt4的调控作用，构建出luciferase-Wnt4的荧光素酶载体，将载体质粒与mimics和 inhibitor共转染至棕色脂肪前体细胞，发现mir24mimics对Wnt4野生型载体有明显抑制作用，而mir24inhibitor对Wnt4野生型载体有明显促进作用，证实了mir-24靶向抑制靶基因Wnt4的mRNA表达。.我们将pCDH-Wnt4共转染至棕色脂肪前体细胞，使Wnt4基因过表达，采用Wnt4-siRNA的方法将细胞Wnt4基因沉默。测定vec对照组、Wnt4过表达组、shRNA对照组和Wnt4表达抑制组的增殖活性，发现Wnt4过表达后的细胞增殖活力有下降趋势；Wnt4基因沉默后细胞增殖活力有上升趋势。油红O 染色结果显示Wnt4过表达促进脂肪细胞分化，而Wnt4表达抑制则抑制脂肪细胞分化，可能与抑制细胞增殖相关。.RT-PCR结果发现Wnt4过表达组与vec对照组相比PPARr、C/EBPβ、aP2均表达增加， PGC-1a表达增加，虽UCP-1和PRDM16表达有增加趋势，但无统计学差异；而Wnt4表达抑制组与对照组相比，PPARr（P值=0.121）、C/EBPβ（P值=0.35）及PGC-1a（P值=0.4） 、UCP-1（P值=0.19）、PRDM16（P值=0.28）的mRNA表达均有下降的趋势，但无统计学差异。.蛋白水平层面结果显示Wnt4过表达组与对照组相比PPARr、C/EBPβ、aP2、PGC-1a、UCP-1和PRDM16的蛋白表达水平显著增加，具有显著统计学差异（P值为0.000，0.003，0.012，0.002, 0.002, 0.000）；而Wnt4表达抑制组与对照组相比，脂肪细胞分化相关的转录因子PPARr、C/EBPβ、aP2、PGC-1a、UCP-1和PRDM16的蛋白表达水平均显著下降，有显著统计学差异（P值均<0.001）。 .我们的研究结果表明，Wnt4通路可促进棕色脂肪细胞分化并抑制细胞增殖，而miR-24通过靶向抑制Wnt4基因对棕色脂肪细胞分化起着负性调节作用。