LncRNA MALAT1 作为ceRNA调控miR-204及其下游因子在角膜新生血管的作用机制研究

Hypoxia is among the major causes of corneal neovascularizaton (NV). Previous studies showed that miR-204 can inhibit corneal NV. Hypoxia can induce the overexpression of lncRNA MALAT1, which can inhibit miR-204 as ceRNA, thus resulting in the up-regulation of the downstream targets of miR-204, including Angpt1 and Amot, which will lead to corneal NV. In this study, we will use hypoxia-induced corneal cells and vascular endothelial cells as in vitro model, and alkali burn-induced mouse corneal NV as in vivo model. Dual-luciferase assay will be used to analyze whether MALAT1 could specificly target miR-204 and thus inhibit its effect. The expression of MALAT1 will be up-regulated and down-regulated by transfecting MALAT1 plasmid and GapmeR/siRNA, and the effects of MALAT1 on cell proliferation, migration, vascular tube formation and mouse corneal NV will be observed. The regulation of MALAT1 on miR-204 and its downstream Amot-YAP-TEAD-VEGF pathway as well as the expression of Angpt1 will be detected from the cellular, protein and mRNA levels. Our aim is to investigate the regulative effect and its molecular mechanism of MALAT1 on miR-204 and its downstream targets, thus providing theoretical basis for the therapeutic development of corneal NV.

缺氧是角膜NV的主要原因。前期已知miR-204有抑制角膜NV作用。缺氧诱导lncRNA MALAT1作为ceRNA靶向抑制miR-204，引发miR-204下游Angpt1和Amot高表达，导致血管新生。本研究用缺氧诱导的角膜细胞和血管内皮细胞为体外模型，碱烧伤诱导的小鼠角膜NV为体内模型，运用dual-luciferase assay分析MALAT1可否靶向结合miR-204并抑制其作用。通过转染MALAT1质粒或GapmeR/siRNA上调或下调MALAT1表达，观察其对细胞增殖、迁移、管腔形成及小鼠角膜NV的影响；运用细胞分子生物学技术，从细胞、蛋白、mRNA水平检测MALAT1、miR-204及其下游Amot-YAP-TEAD-VEGF通路各因子以及Angpt1的动态变化，阐明MALAT1对miR-204及其下游因子的调控作用及分子机制，为治疗角膜NV的新药研发提供理论基础。

缺氧是角膜血管新生的主要原因。前期研究已知miR-204有抑制角膜新生血管形成作用。在此基础上，发现lncRNA MALAT1可以内源性竞争RNA（competing endogenous RNA, ceRNA）形式调控miR-204，推测MALAT1在角膜血管新生过程中可能发挥重要调控作用。.基于此，本课题以缺氧诱导的HUVEC细胞为体外模型，以碱烧伤诱导角膜新生血管小鼠模型为体内模型，运用shMALAT1及pri-miR-204分别抑制MALAT1及上调miR-204表达，结果发现两者均可有效抑制HUVEC细胞增殖及管腔形成以及角膜新生血管形成。说明下调MALAT1表达可有效抑制缺氧诱导的角膜血管新生过程。其次通过双荧光素酶报告基因检测分析，证实lncRNA MALAT1可靶向结合miR-204，作为ceRNA调控miR-204的表达；而miR-204可靶向结合Amot，后者可能为新发现的miR-204靶向调控基因。最后，运用PCR、Western blot技术，分析发现缺氧可诱导MALAT1表达上调，并通过调控miR-204及其下游Amot/VEGF通路以及Angpt1表达在角膜血管新生过程中发挥重要作用。进一步明确MALAT1在角膜血管新生过程中的作用机制：损伤或病变导致的缺氧可诱导角膜组织中lncRNA MALAT1高表达，后者作为ceRNA抑制miR-204作用，引起miR-204下游靶标基因Angpt1和Amot的表达增高，前者是已知促血管形成因子，后者可通过促进VEGF表达激活血管新生过程。本研究结果将为治疗角膜血管新生的新药研发提供理论基础。项目目前已发表论文2篇，已录用论文1篇。