Shaanxi Eye Hospital, Xi’an People’s Hospital (Xi’an Fourth Hospital), Affiliated Guangren Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi 710004, China.
Aging (Albany NY). 2023 Apr 13;15(18):9341-9357. doi: 10.18632/aging.204647.
High glucose promotes retinal pigment epithelial cell (RPEC) migration. However, the underlying molecular mechanisms explaining how high fatty acid levels affect RPEC migration remain largely unknown. We investigated whether and how palmitic acid (PA) impacts the migration of human RPEC cell line ARPE-19. ARPE-19 cells were treated with varying doses of palmitic acid, and the RPEC migration was evaluated by scratch and transwell migration assays. Cell viability was determined by the CCK-8 method. The levels of epithelial-mesenchymal transition (EMT)-associated proteins, including E-cadherin, vimentin, MMP2, and MMP3, were evaluated by western blot. The microRNAs and mRNAs levels were assessed by quantitative PCR. miRNA targets were predicted with online tools and validated with the luciferase reporter assay. miRNA mimics, inhibitors, and siRNA oligos were used to perform gain-of-function and loss-of-function studies. We found that PA increased viability of ARPE-19 cells, promoted their migration and EMT. PA decreased E-cadherin protein expression, and increased vimentin, MMP2, and MMP3 protein levels. Additionally, PA increased miR-222 expression in ARPE-19 cells, and functionally blocking miR-222 suppressed the PA-induced RPEC migration and EMT. NUMB was identified as a downstream target of miR-222, and NUMB knockdown abolished the effects of PA on promoting the migration and EMT of ARPE-19 cells. Therefore, PA promotes human RPEC migration by upregulating miR-222 expression and downregulating NUMB. This study unravels a novel PA-miR-222-NUMB axis that can be potentially targeted for therapy of high fat acid-related ocular diseases.
高葡萄糖促进视网膜色素上皮细胞（RPEC）迁移。然而，高脂肪酸水平如何影响 RPEC 迁移的潜在分子机制在很大程度上仍不清楚。我们研究了棕榈酸（PA）是否以及如何影响人 RPEC 细胞系 ARPE-19 的迁移。用不同剂量的棕榈酸处理 ARPE-19 细胞，通过划痕和 Transwell 迁移实验评估 RPEC 迁移。用 CCK-8 法测定细胞活力。通过 Western blot 测定上皮-间充质转化（EMT）相关蛋白，包括 E-钙粘蛋白、波形蛋白、MMP2 和 MMP3 的水平。用定量 PCR 评估 microRNA 和 mRNA 水平。用在线工具预测 miRNA 靶标，并通过荧光素酶报告基因实验进行验证。用 miRNA 模拟物、抑制剂和 siRNA 寡核苷酸进行功能获得和功能丧失研究。我们发现 PA 增加了 ARPE-19 细胞的活力，促进了它们的迁移和 EMT。PA 降低了 E-钙粘蛋白蛋白表达，增加了波形蛋白、MMP2 和 MMP3 蛋白水平。此外，PA 增加了 ARPE-19 细胞中的 miR-222 表达，功能性阻断 miR-222 抑制了 PA 诱导的 RPEC 迁移和 EMT。NUMB 被鉴定为 miR-222 的下游靶标，NUMB 敲低消除了 PA 对促进 ARPE-19 细胞迁移和 EMT 的作用。因此，PA 通过上调 miR-222 表达和下调 NUMB 促进人 RPEC 迁移。这项研究揭示了一个新的 PA-miR-222-NUMB 轴，可作为治疗与高脂肪酸相关的眼部疾病的潜在靶点。
