Han Xiao-Dong, Jiang Xu-Guang, Yang Min, Chen Wen-Jun, Li Li-Gang
Department of Ocular Fundus Diseases, Xi'an Aier Ancient City Eye Hospital, Xi'an, Shaanxi 710082, P.R. China.
Department of Cataracts, Xi'an Aier Ancient City Eye Hospital, Xi'an, Shaanxi 710082, P.R. China.
Exp Ther Med. 2022 Jun 1;24(1):481. doi: 10.3892/etm.2022.11408. eCollection 2022 Jul.
The present study revealed that palmitic acid (PA) treatment induced epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells, which are involved in the progression of proliferative vitreoretinopathy (PVR). ARPE-19 cells were treated with PA followed by miRNA screening and EMT marker detection using qRT-PCR. Then, miR-124 mimic or inhibitor was transfected into ARPE-19 cells to explore the role of miR-124 on the EMT of ARPE-19 cells using transwell assay. The underlying mechanism of miRNA were predicted by bioinformatics method and confirmed by luciferase activity reporter assay. Furthermore, gain-of-function strategy was also used to explore the role of LIN7C in the EMT of ARPE-19 cells. The expression of miRNA or mRNA expression was determined by qRT-PCR and the protein expression was determined using western blot assay. The result presented that PA reduced the expression of E-cadherin/ZO-1 whilst increasing the expression of fibronectin/α-SMA. In addition, PA treatment enhanced the expression of microRNA (miR)-124 in ARPE-19 cells. Overexpression of miR-124 enhanced PA-induced upregulation of E-cadherin and ZO-1 expression and downregulation of fibronectin and α-SMA. Moreover, miR-124 mimic also enhanced the migration of ARPE-19 cells induced by PA treatment. Inversely, miR-124 inhibitor presented opposite effect on PA-induced EMT and cell migration in ARPE-19 cells. Luciferase activity reporter assay confirmed that Lin-7 homolog C (LIN7C) was a direct target of miR-124 in ARPE-19 cells. Overexpression of LIN7C was found to suppress the migration ability and expression of fibronectin and α-SMA, while increasing expression of E-cadherin and ZO-1; miR-124 mimic abrogated the inhibitive effect of LIN7C on the EMT of ARPE-19 cells and PA further enhanced this abolishment. Collectively, these findings suggest that miR-124/LIN7C can modulate EMT and cell migration in RPE cells, which may have therapeutic implications in the management of PVR diseases.
本研究表明，棕榈酸（PA）处理可诱导视网膜色素上皮（RPE）细胞发生上皮-间质转化（EMT），而这一过程与增殖性玻璃体视网膜病变（PVR）的进展有关。用PA处理ARPE-19细胞，随后进行miRNA筛选，并使用qRT-PCR检测EMT标志物。然后，将miR-124模拟物或抑制剂转染到ARPE-19细胞中，通过Transwell实验探究miR-124对ARPE-19细胞EMT的作用。通过生物信息学方法预测miRNA的潜在机制，并通过荧光素酶活性报告基因实验进行验证。此外，还采用功能获得策略探究LIN7C在ARPE-19细胞EMT中的作用。通过qRT-PCR测定miRNA或mRNA的表达，使用蛋白质印迹法测定蛋白质表达。结果显示，PA降低了E-钙黏蛋白/ZO-1的表达，同时增加了纤连蛋白/α-平滑肌肌动蛋白的表达。此外，PA处理增强了ARPE-19细胞中微小RNA（miR）-124的表达。miR-124的过表达增强了PA诱导的E-钙黏蛋白和ZO-1表达上调以及纤连蛋白和α-平滑肌肌动蛋白表达下调。此外，miR-124模拟物还增强了PA处理诱导的ARPE-19细胞迁移。相反，miR-124抑制剂对PA诱导的ARPE-19细胞EMT和细胞迁移呈现相反的作用。荧光素酶活性报告基因实验证实，Lin-7同源物C（LIN7C）是ARPE-19细胞中miR-124的直接靶点。发现LIN7C的过表达抑制迁移能力以及纤连蛋白和α-平滑肌肌动蛋白的表达，同时增加E-钙黏蛋白和ZO-1的表达；miR-124模拟物消除了LIN7C对ARPE-19细胞EMT的抑制作用，而PA进一步增强了这种消除作用。总的来说，这些发现表明miR-124/LIN7C可以调节RPE细胞中的EMT和细胞迁移，这可能对PVR疾病的治疗具有重要意义。
