miR-204与miR-30a在后发性白内障中对上皮间质转化的网络调控机制

A secondary cataract, also termed as posterior capsular opacification (PCO), is the most common complication of cataract surgery. Following the insult of surgery, residual lens epithelial cells (LECs) rapidly grow at the equator and under the anterior lens capsule. This condition is known as PCO. The remaining LECs undergo an epithelial-to-mesenchymal transition (EMT), resulting in the formation of fibroblasts instead of normal differentiation into lens fiber cells. EMT is the principal pathological change of PCO after lens extracapsular extraction. According to previous studies, miR-30a and miR-204 are the common part of microRNAs differential expression profile of PCO in human, mouse and rat. The predicted target genes conclude Smad4、Smad5、MAPK1、Snail1/2 and ZEB1, which belong to the major signal pathways involved in SMADs pathway, MAPK/ERK Kinase signaling pathway, and transcription factors pathway. All of these pathways are the main and key pathways of EMT. In the previous studies, we also found that miR-204 directly regulates EMT through its targeting of SMAD4. Therefore, in this study we will use capsular bag cultivation model in vitro and PCO animal model in vivo to further investigate the regulational mechanisms of miRNAs in EMT of PCO. We are going to comlpete the differential expression profiling of microRNAs on rat PCO, and to investigate the role of miR-30a and miR-204 in regulating EMT during PCO by using bioinformatics, luciferase reporter gene systerm, Western blot, real-time PCR, immunofluorescence, and siRNA techniques. We hope this study will help to move forward a further step to give a novel insight of molecular mechanisms of PCO and find some novel targets for PCO therapeutic intervention.

晶状体上皮间质转化（EMT）是后发性白内障（PCO）的主要病理改变。课题组前期研究显示：miR-204和miR-30a 是人、小鼠和大鼠PCO组织共同差异表达的miRNA；其预测的靶基因包括Smad4、Smad5、MAPK1、Snail1/2和ZEB1等，涉及了EMT发生的三条主要信号途径，且前期工作证实miR-204及其靶基因Smad4通过Smad途径显著调控PCO中EMT进程。本研究将完善大鼠PCO不同时间miRNA差异表达谱，以miR-30a与miR-204为主要研究对象，结合生物信息学，应用报告基因系统、Western blot、定量PCR、免疫荧光以及干扰RNA等技术，利用囊袋培养和PCO动物模型研究上述miRNAs调控晶状体上皮细胞EMT的作用机制以及通过miRNAs干预PCO的可行性。本项目从转录后水平探讨PCO发病机制，以期为防治PCO提供新的研究思路和干预手段。

晶状体上皮细胞（Lens epithelial cells，LECs）发生上皮-间质转化(Epithelial-mesenchymal transition，EMT) 是导致继发性白内障(posterior capule opcification，PCO) 的主要发病机制之一，建立新的人PCO的体外囊袋培养模型，探讨miRNAs在PCO形成过程中对LECs EMT的发生机制的调控，对于PCO的防治具有重要意义。利用健康供体的尸眼行体外模拟的白内障摘出术，游离晶状体囊袋后用昆虫针固定于培养皿中培养，囊袋培养后3天可见后囊膜周边出现LECs并逐渐向中央区增生,培养后7天LECs 完全覆盖后囊膜,略呈铺路石样外观并出现皱褶，此后随着培养时间的延长皱褶的数量增多并伸长,囊袋张力增强。随培养时间的延长，囊袋上LECs中E-cadherin 的表达强度逐渐下降,而α-SMA和 Vimentin 的表达强度逐渐增强。体外高糖模型中，miR-30a 通过靶作用于SNAIL1对 EMT 相关因子的具有调控作用。本研究中的新囊袋模型中进行miRNAs对LECs EMT的调节，是探讨PCO和糖尿病性白内障方法发生机制新的体外细胞模型。