肥胖相关miR-424在人脂肪间充质干细胞成脂分化中的功能与机制研究

Overexpression of miR-424 was investigated not only in visceral adipose tissue of obese individuals（vs normal weight humans）but also in the mature adipocytes differentiated from human adipose-derived mesenchymal stem cells (hASCs) by using miRNA microarrays. The miR-424 was gradually upregulated during the differentiation of hASCs. Overexpression of miR-424 effectively induced adipogenesis of hASCs. LRP6, a receptor of Wnt signaling, is considered as a candidate target of miR-424 by bioinformatic analysis and initial verification. Based on the previous results, We proposed the hypothesis that miR-424 inhibit the Wnt signaling by downregulate the expression of LRP6 which contribute to the differentiation and adipogenesis of hASCs and involved in obesity. This research will verify the relationship between miR-424 and obesity with larger sample. The function of miR-424 on hASCs differentiation and Wnt signaling will be investigated by overexpression and silence techniques. Furthermore, we will also explore the mechanism of miR-424 on adipogenesis through the Wnt signaling depend on target gene LRP6. miR-424 might be involved in the pathogenesis of obesity which will provided a new potential target in the prevention and treatment of obesity.

miR-424是我们应用芯片筛选获得的miRNA，既差异高表达于肥胖者网膜脂肪组织（Vs正常体重），又高表达于成熟脂肪细胞（Vs人脂肪间充质干细胞）。前期发现，miR-424在人脂肪间充质干细胞成脂分化过程中表达逐渐上调；过表达miR-424可显著促进人脂肪间充质干细胞成脂分化；生物信息学和实验初步提示Wnt信号通路受体LRP6为其靶基因。基于前期结果我们提出“miR-424 通过靶向抑制LRP6 表达，导致Wnt信号传导受抑，进而促进人脂肪间充质干细胞成脂分化、参与肥胖发生”的科学假说。本研究拟：扩大临床样本，以论证miR-424与肥胖之间的关系；通过过表达/沉默策略，论证miR-424在成脂分化中的作用；以LRP6为切入点，探讨miR-424调控人脂肪间充质干细胞成脂分化的分子机制。本研究如获成功，将有助于更深入了解人脂肪间充质干细胞成脂分化分子机制，为肥胖防治提供新的靶标。

脂肪来源间充质干细胞系（hASCs）可定向分化为脂肪细胞或成骨细胞。miR-424是我们应用芯片筛选获得的miRNA, 预测靶基因为Wnt信号通路受体LRP6。通过前期研究，我们提出“miR-424 通过抑制LRP6 表达，导致Wnt信号传导受抑，进而促进hASCs成脂分化、参与肥胖发生”的科学假说。.为此，本研究首先通过收集儿童非肥胖手术患者皮下及网膜脂肪组织，比较前期筛选的miRNA部位表达差异特征，分析miRNA与生长发育相关指标、血生化指标的关系；进一步分析miR-424与脂肪分化标志基因及Wnt信号通路受体基因的相关性；通过在皮下脂肪干细胞（SC-ASCs）中过表达及沉默策略，结合萤光素酶报告基因检测及挽救实验，论证 miR-424通过靶基因LRP6调控SC-ASCs成脂分化。.研究结果表明：1.腹部皮下脂肪组织miR-424表达显著高于网膜，且皮下miR-424与LRP6呈显著负相关；2、SC-ASCs中miR-424过表达促进SC-ASCs向脂肪细胞分化、成熟，脂肪分化关键基因PPARγ、C/EBPα表达明显增高，而LRP6表达及蛋白水平下降；相反，miR-424沉默后，SC-ASCs向脂肪分化能力减弱，而LRP6表达量反而增高；3、LRP6过表达抑制SC-ASCs向脂肪细胞分化，荧光素酶报告基因检测实验验证miR-424可与LRP6 的3’UTR结合，而突变后的LRP6 其3’UTR不能与miR-424结合，证明LRP6为miR-424的靶基因。.通过本项目资助，发现miR-424通过抑制靶基因LRP6表达，减弱Wnt信号通路活性，从而促进SC-ASCs向脂肪细胞分化、成熟，可能是儿童腹部皮下脂肪组织增多的重要机制之一。