Jun Jong Hwa, Joo Choun-Ki
Department of Ophthalmology, Keimyung University School of Medicine, Dongsan Medical Center, Daegu, Korea.
Department of Ophthalmology and Visual Science, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea 3Catholic Institute for Visual Science, The Catholic University of Korea, College of Medicine, Seoul, Korea.
Invest Ophthalmol Vis Sci. 2016 Jan 1;57(1):12-22. doi: 10.1167/iovs.15-17111.
MicroRNA-124 (miR-124) is thought to be involved in the epithelial-mesenchymal transition (EMT) of RPE. We investigated the regulation of TGF-β1-induced EMT by miR-124 in human RPE cells (ARPE-19).
Expression of miR-124 was evaluated after TGF-β1 treatment by quantitative RT-PCR. Phenotypic alterations were analyzed by Western blot analysis and immunocytochemical staining. Target validation was performed by a luciferase reporter assay to identify the putative target of miR-124.
The expression level of miR-124 was downregulated during the progression of EMT. Overexpression of miR-124 upregulated the levels of zonular occludens 1 and occludin, and downregulated those of fibronectin, α-smooth muscle actin, and vimentin. Furthermore, inhibition of endogenous miR-124 increased and decreased the levels of mesenchymal and epithelial factors, respectively. TargetScan predicted two well-conserved and two vertebrate-only conserved miR-124 target sequences in the 3' untranslated region (UTR) of the Ras homology Growth-related (RHOG) mRNA. Direct targeting of this 3' UTR by miR-124 was demonstrated using a luciferase assay. Silencing of RHOG using a specific siRNA had identical effects on EMT regulation. Overexpression of miR-124 repressed TGF-β1-induced RPE cell-collagen gel lattice contraction by altering cell spreading/cell-to-cell adhesion.
This study describes the regulation of EMT in RPE cells by TGF-β1/miR-124/RHOG signaling and suggests that the supplement of miR-124 exogenously would be a valuable therapeutic approach for the prevention or treatment of proliferative vitreoretinopathy.
微小RNA-124（miR-124）被认为参与视网膜色素上皮（RPE）的上皮-间质转化（EMT）过程。我们研究了miR-124对人RPE细胞（ARPE-19）中转化生长因子-β1（TGF-β1）诱导的EMT的调控作用。
通过定量逆转录聚合酶链反应（qRT-PCR）评估TGF-β1处理后miR-124的表达。通过蛋白质免疫印迹分析和免疫细胞化学染色分析表型改变。通过荧光素酶报告基因检测进行靶标验证，以鉴定miR-124的假定靶标。
在EMT进程中，miR-124的表达水平下调。miR-124的过表达上调了紧密连接蛋白1和闭合蛋白的水平，并下调了纤连蛋白、α-平滑肌肌动蛋白和波形蛋白的水平。此外，抑制内源性miR-124分别增加和降低了间充质和上皮因子的水平。TargetScan预测在Ras同源生长相关蛋白（RHOG）mRNA的3'非翻译区（UTR）中有两个高度保守和两个仅在脊椎动物中保守的miR-124靶序列。使用荧光素酶检测证明miR-124直接靶向该3'UTR。使用特异性小干扰RNA（siRNA）沉默RHOG对EMT调控具有相同的作用。miR-124的过表达通过改变细胞铺展/细胞间粘附来抑制TGF-β1诱导的RPE细胞-胶原凝胶晶格收缩。
本研究描述了TGF-β1/miR-124/RHOG信号通路对RPE细胞中EMT的调控，并表明外源性补充miR-124可能是预防或治疗增殖性玻璃体视网膜病变的一种有价值的治疗方法。
