LncRNA-LINC01705通过miR-29b促进视网膜色素上皮-间充质转换的作用及机制

Proliferative vitreoretinopathy (PVR) is a severe blinding disease.Currently, there are no effective early therapy or prevention strategies for PVR. Epithelial-mesenchymal transition of retinal pigment epithelial (RPE) is a hallmark in the initiation and development of PVR. We previously reported that down-regulation of miR-29b is one of the key mechanisms of EMT in RPE. Current studies revealed the role of lncRNA in the EMT of various cancer cells, while its role in the EMT of RPE or in the development of PVR is not clear. By using lncRNA microarray and RT-PCR, we previous identified a potential lncRNA-LINC01705 which may play a significant role in the EMT process of RPE probably through regulating miR-29b. In this project, we will explore the role, mechanism, and clinical association of LINC01705 in PVR with the EMT model of primary human RPE, rabbit PVR model, and clinical samples and materials. Experimental techniques like RT-PCR, western blot, immunofluorescence, flourescence in situ hybridization, RNA immunoprecipitation, chromotin immunoprecipitation will be used in this project. The completion of this project will not only provide a more comprehensive understanding for the mechanisms of PVR, but also solidify the theoretic foundation for the early diagnosis, treatment and prognosis of PVR.

增生性玻璃体视网膜病变（PVR）是一种严重的致盲性眼病。目前无有效预防和早期治疗方法。视网膜色素上皮（RPE）发生上皮-间充质转换（EMT）是PVR启动的关键，我们前期研究证实miR-29b下调是RPE发生EMT的关键机制之一。研究表明长链非编码RNA（lncRNA）可调控肿瘤细胞EMT，而其在RPE细胞EMT及PVR中的作用仍不明确。本研究前期利用lncRNA芯片联合RT-PCR筛查出在RPE细胞EMT中的潜在关键性lncRNA-LINC01705，预测其可能通过下调miR-29b发挥作用。本项目将进一步在RPE细胞EMT模型、PVR动物模型、及临床资料中，采用RT-PCR、免疫印迹、免疫荧光、荧光原位杂交、RNA免疫沉淀、染色质免疫沉淀等方法对LINC01705在PVR中的功能、机制及临床价值展开深入研究。研究成果有助于更全面理解PVR发病机制，为PVR早期诊治、预后评估奠定理论基础。

增生性玻璃体视网膜病(PVR)是一种严重的致盲性眼病，其特征是在视网膜下或视网膜前形成收缩性纤维化增殖膜。视网膜色素上皮(RPE)细胞上皮-间充质转化(EMT)是PVR的标志。本项目探讨了长链非编码RNA (lncRNA)-LINC01705在RPE中的表达及其在PVR中的作用，并探讨其潜在的机制。我们将该LncRNA命名为ERLR(EMT-related lncRNA in RPE). 在本研究中，我们通过lncRNA芯片和RT-PCR检测到，在转化生长因子(TGF)-β1刺激的RPE细胞中ERLR上调。进一步的研究鉴定了其全长碱基序列，并证实其主要表达在细胞质中。在体外实验中，在RPE细胞中沉默ERLR可抑制TGF-β1诱导的EMT，而过表达ERLR可直接触发RPE细胞的EMT。 在体内，抑制RPE细胞的ERLR会降低细胞诱导实验性PVR的能力。机制上，染色质免疫沉淀(ChIP)检测表明转录因子TCF4直接结合到ERLR的启动子区并促进其转录。ERLR通过直接结合MYH9蛋白并增加其稳定性来介导EMT。TCF4和MYH9也可直接介导TGF-β1诱导的RPE细胞EMT。此外，与临床PVR玻璃体手术标本孵育的RPE细胞的ERLR也显著增加。在PVR膜的临床样本中，通过荧光原位杂交(FISH)检测ERLR，并与RPE标记的全细胞角蛋白(pan-CK)共定位。 提示lncRNA ERLR参与TGF-β1诱导的人RPE细胞EMT，参与PVR。这一发现为PVR的机制和治疗提供了新的见解。

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