HuR介导lncRNA HOXA11-AS1调控脂肪细胞分化及肥胖症发病的分子机制

Numerous studies have identified transcription factor PPARγ and fatty acid binding protein（FABP4）directly involved in obesity. In recent years, it was reported that long chain non-coding RNA (lncRNA) and human antigen R (HuR) were associated with adipocyte differentiation and obesity , but the mechanism remains unknown. In the preliminary study ,we found that lncRNA HOXA11-AS1、HuR and FABP4 expression increase significantly during adipocyte differentiation. Not only the lncRNA gene promoter region has PPARγ combination motif but also the lncRNA combining with HuR is relatively high level, while HuR combined with FABP4 simultaneously. However, it is unclear that interaction between the three molecules and its function in the adipocyte differentiation. This study aims at determining the PPARγ promoter region combined by the lncRNA gene and its regulatory role. Furthermore we will discuss the molecular mechanism that the lncRNA coupling HuR complexes raised FABP4 expression level by using the molecular biology techniques and adipocyte differentiation model. Finally we will use clinical samples to verify the above experimental results in vitro. This study will clarify lncRNA HOXA11-AS1 role in the adipocyte differentiation and obesity and its molecular mechanism, and so with high innovative and medical significance.

大量研究已确定转录因子PPARγ和脂肪酸结合蛋白4（FABP4）直接参与肥胖症发病。近年报道长链非编码RNA（lncRNA）和人类抗原R（HuR）与脂肪细胞分化及肥胖症有关，但机制不明。我们前期研究发现脂肪细胞分化时lncRNA HOXA11-AS1、HuR、FABP4表达显著升高，该lncRNA 基因启动子区有PPARγ结合基序且与HuR有较高结合丰度，HuR也可与FABP4结合，但三者之间的相互作用及其在脂肪细胞分化中的功能尚不清楚。本项目拟采用ChIP、RIP等分子生物学技术和人脂肪细胞分化模型，确定PPARγ与该lncRNA基因启动子区的结合及调节作用，探讨该lncRNA与HuR形成的复合物在转录后水平上调FABP4表达的机制，并采用临床样本验证上述体外实验结果，以阐明lncRNA HOXA11-AS1在脂肪细胞分化及肥胖症发病过程中的作用及其分子机制，具有较高创新性及医学意义。

肥胖是2型糖尿病、高血压、心脑血管疾病等的高危因素，肥胖发生机制的研究具有重要的临床意义。脂肪细胞异常分化导致的脂肪细胞数量增多或体积增大都会引起脂肪组织的增多进而导致肥胖。因此，研究脂肪细胞分化的调控机制对探讨肥胖的发生具有重要的意义。本课题采用RT-qPCR方法检测hADSCs中lncRNA HOXA11-AS1的表达，通过敲低和过表达实验分析了lncRNA HOXA11-AS1对hADSC脂肪形成的影响，RNA-IP和RNA pull down实验确认HuR在FABP4和lncRNA HOXA11-AS1上的结合。通过免疫荧光和核质分离实验确认lncRNA HOXA11-AS1对HuR细胞定位的影响，而放线菌素D实验确定HuR对FABP4 mRNA稳定性的影响。结果显示，lncRNA HOXA11-AS1受PPARγ转录调控，上调脂肪细胞分化相关基因FABP4，CEBP-α，ACC，FASN的基因表达水平，增加脂肪细胞内脂质聚集，进而促进脂肪细胞分化。在脂肪细胞分化过程中lncRNA HOXA11-AS1结合RNA结合蛋白HuR，促进HuR由细胞核向细胞质转位，增加HuR与FABP4的结合率，进而使FABP4 mRNA稳定性增加，上调FABP4的蛋白质表达水平。临床样本结果显示，lncRNA HOXA11-AS1、FABP4、CEBP-α、FASN的mRNA表达水平在肥胖组中显著升高，且lncRNA HOXA11-AS1表达水平与臀围以及脂肪细胞分化相关基因FABP4、CEBP-α、FASN、ACC的表达呈正相关，提示lncRNA HOXA11-AS1与肥胖相关。本课题在建立人脂肪细胞分化模型的基础上，通过研究lncRNA HOXA11-AS1在脂肪细胞分化过程中的功能以及与HuR共同作用在转录后和翻译水平对FABP4蛋白质表达水平的调控作用，揭示lncRNA HOXA11-AS1促进脂肪细胞分化的机制，为今后人们对肥胖的认识、预防和治疗提供重要的立论依据。