基因间长链非编码RNA-p21在年龄相关性白内障中的作用及调控机制研究

Our preliminary study indicated that long intergenic non-coding RNA p21（lincRNA-p21）was up-regulated in age-related cataract（ARC）, knockdown of lincRNA-p21 reduced acetylated p53 and p53 target genes. It is predicted by bioinformatics that lincRNA-p21 binds to the RING domain of MDM2, and we proposed that this interaction may diminish the inhibition of MDM2/p53 compound to acetylated p53 induced by p300, such enhanced the acetylated p53 and the transcriptional activity of p53 to regulate the proliferation, apoptosis, endoplasmic reticulum stress and autophagy of lens epithelial cells (LECs) in ARC. This research intends to build over expression or low expression vector, by methods of RNA-Seq, RNA-pulldown, RIP, ChIP and Dual- luciferase to verify the direct interaction of p53 and MDM2 and the network regulation mechanism; as well as to determine the effect of the mechanism above in p53CKO mice. We hope this research to afford a new target for the prevention and treatment of ARC.

项目组前期工作发现lincRNA-p21在年龄相关性白内障（ARC）中显著高表达，敲减lincRNA-p21后，lys-382乙酰化p53蛋白水平表达减低，p53下游靶基因表达下调。生物信息学预测lincRNA-p21与MDM2蛋白RING结构域直接结合，我们推测这种互作可解除MDM2/p53复合体对p300介导的p53乙酰化的抑制，提高乙酰化p53水平，增强p53转录激活活性，实现对ARC发生发展过程中晶状体上皮细胞增殖凋亡、内质网应激及自噬等生物学过程的调控。我们将通过构建lincRNA-p21过表达或低表达载体，应用RNA-Seq、RNA-pulldown、RIP、ChIP、Dual-Luciferase等方法，阐明lincRNA-p21与MDM2直接结合的机制，并通过p53CKO小鼠证实lincRNA-p21在体内对ARC发生发展的调控机制，为ARC的防治提供新靶点。

白内障是世界上首要的致盲眼病，全世界范围内有2千万人因白内障而致盲，年龄相关性白内障（Age-related cataract，ARC）是其中最常见的一类，占比达50%以上。本研究拟以人晶体上皮细胞（ HLE-B3 ）为研究对象，通过过氧化氢体外构建氧化损伤模型，利用活性氧试剂盒检测细胞模型中ROS的变化，流式和免疫荧光等方法检测细胞增殖相关指标（细胞周期和EDU）的变化。流式检测细胞凋亡、Western Blot方法检测凋亡相关指标（Bax，Bcl-2），Caspase-3 活性检测试剂盒检测细胞凋亡蛋白 Caspase-3的活性。利用RT-qPCR检测细胞模型中lncRNA-p21的表达，FISH检测lncRNA-P21的细胞定位，以及检测P53蛋白和RNA水平变化，结果发现在年龄相关性白内障细胞模型中细胞增殖率显著降低、凋亡率显著上升，ROS表达水平升高，P53的 mRNA 相对表达量明显升高，这些变化可能与lncRNA-p21水平升高密切相关。此外，我们还探究了内质网应激对晶状体上皮细胞增殖、凋亡的影响，并行转录组学检测，进一步对其机制进行研究，对延缓和阻止ARC的发生发展，具有重要及深远的科学意义、社会意义及临床应用前景。