衰老相关LncRNA ANRIL及PINT参与的氧化损伤清除基因GST的表观遗传调控在年龄相关性白内障发病中的作用机制研究

The etiology and the exact pathogenesis of the age-related cataract (ARC) remain unclear. Our previous study shows that the epigenetic changes of glutathione S transferase (GST) are associated with ARC formation, but the mechanism of epigenetic changes has not been investigated. Recently, we have observed that there is differential expression of senescence-associated LncRNA ANRIL (antisense non-coding RNA in the INK4 locus) and PINT (p53 induced noncoding transcript) in ARC lens. We hypothesize that ANRIL and PINT can regulate the epigenetic changes of GST by modulating Rb/p16 and p53/p21 pathways during the ARC formation. In current study, we have collected different type of ARC lens and transparent lens to examine the expression of senescence-associated LncRNA. We have also overexpressed ANRIL and PINT in the human lens epithelial cell lines, and we will knockdown and knockout ANRIL and PINT in the same cell lines to investigate whether ANRIL and PINT are related to apoptosis. Flow cytometry will be used to examine the cell cycle. Oxidative damage and aging model both in vitro and in vivo will be constructed to explore whether ANRIL and PINT are involved in oxidative damage and aging. The FISH (fluorescence in situ hybridization), RNA pull-down, mass spectrometry analysis and RNA Binding Protein Immunoprecipitation (RIP) will be used to explore the regulatory mechanism. The study will give us a new insight to elucidate the relationship between the senescence-associated LncRNA and ARC formation and to investigate the pathogenesis of the ARC in epigenetics levels. It will open a new path for early diagnosis of ARC and help to develop potential therapeutic drugs for ARC treatment.

年龄相关性白内障（ARC）病因及确切发病机制尚未探明。我们研究发现氧化损伤清除基因GST表观遗传学改变DNA甲基化、组蛋白修饰与ARC发生相关（已发表），但尚未探讨影响该表观遗传改变的机制。我们预实验发现，衰老相关LncRNA ANRIL及PINT在ARC晶状体中表达降低；结合文献报道提出假设：ANRIL及PINT通过衰老相关Rb/p16和p53/p21通路调控GST表观遗传改变，参与ARC发生。已收集不同类型ARC及透明晶状体，测定衰老相关LncRNA表达；进一步完善体内/体外ANRIL及PINT过表达、Crispr/Cas9模型，观察细胞凋亡、细胞周期；构建氧化损伤/衰老模型，测定抗氧化能力；FISH、RNA pull-down、质谱分析、RIP等探究通路调控机制。揭示衰老相关LncRNA与ARC的关系，从表观遗传学水平探讨ARC发病机制，为ARC防治提供新思路，有助研发抗ARC药物。

年龄相关性白内障（ARC）病因及确切发病机制尚未探明。目前对ARC发生发展的具体分子机制仍缺乏全面了解，阐述某些新基因在该过程中所扮演的角色，对进一步揭示该过程的分子机制有着不可忽视的意义。本研究通过收集不同类型ARC及透明晶状体的上皮细胞，测定衰老相关LncRNA的表达，通过对比ARC组和对照组，找出了差异表达的衰老相关LncRNA PINT（p53 induced noncoding transcript）。PINT的功能依赖于多梳抑制复合物2（polycomb repressive complex 2，PRC2）的表达水平，并通过与PRC2相互作用调控TGF-β、MAPK和p53细胞通路中的蛋白质表达水平。调控表观遗传学变异的LncRNA PINT在p53/p21信号通路中扮演重要角色：p53能通过诱导LncRNA PINT的表达，与PRC2相互作用，进而终止基因转录；调控p53信号通路中的蛋白质表达水平，调控细胞周期进程，从而影响机体的衰老和衰老性疾病的形成。根据基因测序结果，我们用GSEA方法针对KEGG信号通路数据库中的数据进行分析，并通过验证发现，衰老相关LncRNA PINT可能通过招募p53/p21信号通路上的EZH2（enhancer of zeste 2 polycomb repressive complex 2 subunit）调节相关基因的表达，在年龄相关性白内障的发病机制中起到重要作用。EZH2是唯一分布在细胞核中的功能蛋白，作为PRC2的一部分，与LINC-PINT结合，甲基转移酶EZH2对组蛋白H3上赖氨酸27的三甲基化（H3K27me3）作用是基因沉默最重要的表观遗传机制之一。EZH2通过调节p53/p21通路参与细胞凋亡，PINT可能导致GST相关基因表达改变，从而改变人晶状体上皮细胞的抗氧化损伤能力。本研究将衰老相关LncRNA PINT调控氧化损伤清除基因GST相关基因的表达作为切入点，初步阐明了LncRNA PINT可能通过衰老相关p53/p21通路调控相关基因的表观遗传改变，参与ARC的发生发展。揭示了衰老相关LncRNA在年龄相关性白内障的发病机制中的重要作用，可作为ARC防治的新靶点。在表观遗传学水平探索ARC发病机理，为ARC的防治提供新思路，有助研发抗ARC药物，为进一步了解白内障的发病机理提供了思路和方向。