LncRNA-MALAT1作为ceRNA参与miR-204-5p网络调控后发性白内障的分子机制研究

Posterior capsule opacification (PCO) is a common postoperative complication of cataract surgery, and mechanisms of PCO were widely associated with epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs). However, Our preliminary study indicated that PCO is also related with autophagy inhibition. The expression of LncRNA-MALAT1 was up-regulated and the expression of miR-204-5p was down-regulated in human PCO attached LECs, and the predicted target gene Snail, Smad4 and Bcl-2 were up-regulated. Therefore, we proposed our hypothesis that MALAT1 might act as competing endogenous RNAs to regulate the proliferation, migration, EMT and autophagy of LECs by controlling miR-204-5p. This research intends to build over expression or low expression vector and luciferase reporter vector, to verify MALAT1/miR-204-5p/Snail, MALAT1/miR-204-5p/Smad4, MALAT1/miR-204-5p/Bcl-2 axis mediating network regulation mechanisms of EMT and autophagy, and also to verify the biological function; as well as to determine the effect of the signaling axes above on posterior capsule opacification in rat model. We hope this research to afford a new target for the prevention and treatment of PCO.

后发性白内障（PCO）是白内障术后常见并发症，多认为与晶状体上皮细胞（LECs）发生上皮-间质转化（EMT）有关，本组前期研究发现PCO的发生还与自噬受到抑制有关。PCO后囊膜标本中（健康囊膜对照）LncRNA-MALAT1高表达、miR-204-5p低表达、经生物信息学预测的靶基因Snail、Smad4、Bcl-2高表达。因此，我们推测MALAT1可能作为ceRNA通过miR-204-5p调控LECs的增殖、移行、EMT及自噬。我们将构建MALAT1过表达或低表达载体、荧光素酶报告基因载体，阐明PCO形成中MALAT1/miR-204-5p/Snail、MALAT1/miR-204-5p/Smad4、MALAT1/miR-204-5p/Bcl-2轴介导EMT及自噬的网络调控机制，并验证其生物学功能，通过大鼠PCO模型证实上述信号通路轴对后囊膜混浊的影响，为PCO的防治提供新靶点。

白内障在全世界致盲眼病中居首位，据统计术后1-5年内后发性白内障（PCO）成人的发病率为11%-50%，在儿童更是高达100%，PCO已成为影响术后视力的最主要原因。目前认为PCO的发生机制主要认为是晶状体上皮细胞（LECs）发生迁移及上皮-间质转化（EMT）的病理改变，这一过程主要涉及Smads依赖通路及非Smads依赖通路。.MALAT1是一种在多种哺乳动物中呈高丰度表达并且在进化中高度保守的lncRNA，通过ERK/MAPK、Wnt/β-catenin、PI3K/Akt等通路的激活在上皮-间质转化中有重要调控作用。但MALAT1在眼部疾病，特别是包括的晶状体疾病中的研究，目前国内外尚少见明确报道。本项目成功构建了10ng/mL TGF-β2诱导SRA01/04 PCO模型中，并发现MALAT1表达量于TGF-β2诱导24h时出现峰值。又通过FISH RNA技术在国内上首次发现和报道MALAT1定位于SRA01/04细胞的细胞质。经Si-RNA敲减MALAT1，验证敲减效率后，证实Si-MALAT1可抑制TGF-β2诱导的上皮-间质转化，同时SRA01/04细胞的增殖、移行能力减弱。.发现经TGF-β2诱导的SRA01/04细胞PCO模型中，MALAT1与Smad4成正相关表达上调、microRNA-204-5p表达下调。microRNA-204-5p 抑制TGF-β2诱导的SRA01/04细胞上皮-间质转化及增殖、移行。双荧光素酶报告基因实验证实microRNA-204-5p是MALAT1的靶基因，Smad4是microRNA-204-5p直接的靶基因。MALAT1作为ceRNA通过竞争性抑制miRNA-204-5p调控Smad4表达，进而影响TGF-β2诱导的SRA01/04细胞上皮-间质转化。.本项目还发现敲减MALAT1可抑制上皮-间质转化相关信号通路的关键转录因子ZEB1。MALAT1可通过调控TGF-β/Samds、Notch信号通路影响SRA01/04细胞的增殖、移行及上皮-间质转化。过表达ZEB1激动剂可激活TGF-β/Samds、Notch信号通路，并逆转Si-MALAT1对SRA01/04增殖、移行及EMT的影响。.当前转化医学成为热点话题，以MALAT1为代表的lncRNA，可能为临床治疗提供了新思路及可行的PCO防治靶点。