MicroRNA-34a靶向Notchl在后发性白内障中对上皮-间充质转化的调控机制

Posterior capsular opacification (PCO) is a major complication of cataract surgery. epithelial-to-mesenchymal transition (EMT) is one of the most important mechanism of PCO. Our previous study havefound that TGFβ/Smad2、3 and Wnt pathway both involve in the development of EMT as well as the proliferation and migration of lens epithelial cell(LEP). Also, we have found that microRNA(miR)-181/26b regulate above cell functions of LEP. Our preliminary data indicated that both miR-34a and Notch involved in mediating EMT development. By using microRNA and target gene database prediction, we find 3-UTR of Notch have the binding site for miR-34a, and could be the target gene of miR-34a.Therefor,we proposed our hypothesis which is miR-34a targets Notch and regulates EMT of LEP during human PCO after cataract surgery. In this research, we plan to study the following aspects: 1. To examine the expression of miR-34a and Notch in the samples between normal lens and cataract from surgery. 2. To explore the role of miR-34a-Notch in LEP function including proliferation, migration, and EMT by making constructor with specially expressed miR-34 or Notch1, as well as them inhibitor. 3. To study the biding site of miR-34 and Notch1 by using luciferase reporter assays. 4. To determine the effect of miR-34a-Notch on mediating the development of PCO in vivo. This study is useful to determine the mechanism of PCO development, and will afford a novel therapeutical benefit in controlling PCO development after cataract surgery.

后囊混浊(PCO)是白内障术后常见并发症,上皮-间充质转化(EMT)是PCO形成重要机制,我们既往研究显示TGFβ/Smad2、3及Wnt等路径分别调控晶状体上皮细胞(LEP)迁移,增殖和EMT;miR-181a/26b调控LEP上述细胞特性改变.我们预探索表明miR-34a及Notch通路皆与EMT相关.基因组数据库预测miR-34a可能靶向调控Notch1基因,故提出假设miR-34a-Notch信号通路调节后发性白内障的形成.拟研究miR-34a-Notch通路相关基因在白内障手术标本中的表达;构建慢病毒介导的miR-34a及Notch1基因重组体,研究miR-34a-Notch对LEP发生EMT等细胞功能的影响;萤光素酶实验检测miR-34a与Notch1等靶基因的结合位点.研究动物模型中miR-34a-Notch在PCO发生中的作用,为明确PCO形成机制及其控制靶点提供新思路.

后囊混浊(PCO)是白内障术后常见并发症，上皮-间充质转化(EMT)是PCO形成重要机制，我们既往研究显示TGFβ/Smad2、3及Wnt等路径分别调控晶状体上皮细胞(LEC)迁移,增殖和EMT；miR-181a/26b调控LEP上述细胞特性改变。我们研究表明miR-34a及Notch通路皆与EMT相关。基因组数据库预测miR-34a可靶向调控Notch1基因，故提出假设miR-34a-Notch信号通路调节后发性白内障的形成。研究中首先研究miR-34a-Notch通路相关基因在白内障手术标本中的表达。我们研究发现临床标本中miR-34a在混浊的晶状体囊膜中低表达，相反Notch1高表达，同时EMT特征如Fibronectin, Collagen I在混浊的晶状体囊膜高表达；E-cadherin低表达。第二步，我们研究miR-34a靶向调控Notch的分子机制，通过荧光素酶报告实验，我们证实miR-34a在Notch1的3’-UTR区域的165-187结合位点为其调控的关键靶点。在通过TGFb2诱导LEC发生EMT转化模型中，我们同样发现miR-34a在LEC发生EMT中低表达，而Notch1高表达。LEC发生EMT表现典型的高表达如aSMA、fibronectin、collagenI、Vimentin、Snail；低表达E-cadherin。同时Notch1配位体Jagged1表达与Notch1表达一直。通过转染技术，我们过表达miR-34a于晶状体上皮细胞，显著抑制TGF b2诱导的LEC发生EMT转化。另过表达miR-34a显著抑制Notch1表达，运用Notch1抑制剂DAPT可逆转过表达miR-34a的作用，显示了miR-34a通过靶基因Notch1调控晶状体上皮细胞EMT转化。第三步我们运用腺相关病毒构建miR-34a的重组体，探究miR-34a-Notch在抑制后发性白内障模型中的作用。为明确PCO形成机制及其控制靶点提供新思路。