miR-148a多靶点调控Wnt信号通路促进脂肪细胞分化的机制研究

miR-148a is a miRNA expressed highly in human mature adipocyts identified by miRNAs screening chips. Our previous study found that expression of miR-148a increased with the adipocyts differentiation process, and miR-148a highly expressed in adipose tissue of obese children, and miR-148a overexpression could both promote preadipocyts differentiate to mature adipocytes and incrased the accumulation of triglyceride.Moreover, the results from bioinformatics analysis and preliminary experiment showed miR-148a could modulate wnt signal pathway by multiple targets. The study will verify the relationship between miR-148a and obesity. We will establish adipocytes which differentially express miR-148a by overexpression and knock down strategies to explore the function of miR-148a in adipocytes differentiation. Then we will use rescue experiment strategy to analyze the downstream regulation mechanism of miR-148a modulating wnt signal pathway by multiple targets and explore the upstream regulation mechanism of CREB regulating miR-148a transcription. Furthmore, we will evaluate the function and mechanism of miR-148a in vivo by establishing fat-specific transgenic mice. The study will help us understand the mechanism of adipocytes differentiation and obesity development better, and also provide new clue and potential intervention target of control obesity.

miR-148a是本研究团队前期应用miRNA芯片筛选到的一条在人成熟脂肪细胞中差异高表达的miRNA。前期发现，miR-148a随人脂肪细胞分化进程逐渐上调；miR-148a高表达于肥胖儿童脂肪组织；过表达可显著促进脂肪细胞的分化成熟及甘油三酯的积聚；生物信息学及实验均证实miR-148a可多靶点调控wnt信号通路。研究拟进一步论证miR-148a 与肥胖关系；通过过表达、沉默的策略，从正反两方面论证miR-148a 在脂肪分化中的作用；通过挽救实验策略，论证miR-148a 通过多靶点调控Wnt 信号通路影响脂肪细胞分化中的下游调控机制；通过CHIP、过表达等策略，探讨CREB 调控miR-148a 转录的上游调控机制；并通过脂肪特异性转基因小鼠，在体评价miR-148a 的功能与机制。本研究将有助于对脂肪细胞分化及肥胖发生发展机制的理解，并可能为肥胖及其并发症防治提供新线索、新靶标。

miR-148a是本研究小组前期筛选人脂肪间充质干细胞及成熟脂肪细胞的miRNA差异表达谱发现的一个肥胖相关miRNA，在本研究中通过检测不同肥胖生物中miR-148a的表达，发现miR-148a与肥胖密切相关，主要表现为在肥胖者脂肪组织、分化成熟脂肪细胞中高表达。通过功能及机制研究发现：1）miR-148a过表达可促进HMSC-Ad成脂分化；2） miR-148a在转录后水平调控靶基因Wnt1的表达，从而抑制经典Wnt信号通路活性，并阻断Wnt1对HMSC-Ad成脂分化的抑制作用，提示miR-148a是促进HMSC-Ad成脂分化的上游调控因素，并非是分化产生的效应。进一步通过启动子分析及EMSA证实了CREB、E2F可结合至miR-148a启动子区域（-2947 to -2687 nt）， 同时ChIP-qPCR检测发现含有CREB结合位点的区域（-2947 to -2687 nt）IP表达量是input的320倍；而HMSC-Ad细胞中过表达CREB，miR-148a表达水平较对照组明显增加，反之，HMSC-Ad细胞中沉默CREB，miR-148a表达水平较对照组显著降低，提示CREB结合至miR-148a启动子区域，调控了miR-148a的表达， miR-148a是CREB依赖的miRNA。通过本研究基本阐明了miR-148a下游分子途径及上游转录调控机制，有望为肥胖及其并发症防治提供新线索及潜在的干预靶标。