Department of Ophthalmology, Duke University, Durham, North Carolina, USA.
Invest Ophthalmol Vis Sci. 2011 May 6;52(6):2999-3007. doi: 10.1167/iovs.10-6708.
To investigate the biological functions of miR-204 in human trabecular meshwork (HTM) cells.
Changes in gene expression induced by miR-204 in HTM cells were evaluated by gene array analysis using arrays and confirmed by quantitative-PCR (Q-PCR). Direct targeting of miR-204 to 12 potential novel targets was confirmed using a luciferase system, and five of them were verified by Western blot analysis. Effects of miR-204 on apoptosis, cell viability, and accumulation of carbonylated proteins were evaluated in HTM cells treated with H(2)O(2). Induction of endoplasmic reticulum (ER) stress markers by tunicamycin was analyzed by Q-PCR, and expression of IL-8 and IL-11 was analyzed by ELISA.
MiR-204 decreased the expression of multiple genes in HTM cells. Twelve genes (AP1S2, Bcl2l2, BIRC2, EDEM1, EZR, FZD1, M6PR, RAB22A, RAB40B, SERP1, TCF12, and TCF4) were validated as direct targets of miR-204. Downregulation of expressions at protein levels of Bcl2l2, BIRC2, EZR, M6PR, and SERP1 were confirmed by Western blot analysis. HTM cells transfected with miR-204 showed increased levels of apoptosis, decreased viability, increased accumulation of oxidized proteins after H(2)O(2) treatment, decreased induction of ER stress response markers, and reduced expression of inflammatory mediators IL-8 and IL-11.
MiR-204 potentially plays an important role in the regulation of multiple functions in HTM cells including apoptosis, accumulation of damaged proteins, ER stress response, and expression of inflammatory mediators.
研究 miR-204 在人眼小梁细胞（HTM）中的生物学功能。
通过基因芯片分析和定量-PCR（Q-PCR）检测 miR-204 诱导 HTM 细胞基因表达的变化。利用荧光素酶系统证实 miR-204 对 12 个潜在新靶标的直接靶向作用，并通过 Western blot 分析验证其中 5 个靶标。在 HTM 细胞中用 H(2)O(2)处理，评估 miR-204 对细胞凋亡、细胞活力和羰基化蛋白积累的影响。通过 Q-PCR 分析衣霉素诱导的内质网（ER）应激标记物的表达，通过 ELISA 分析 IL-8 和 IL-11 的表达。
miR-204 降低了 HTM 细胞中多个基因的表达。12 个基因（AP1S2、Bcl2l2、BIRC2、EDM1、EZR、FZD1、M6PR、RAB22A、RAB40B、SERP1、TCF12 和 TCF4）被验证为 miR-204 的直接靶标。Western blot 分析证实了 Bcl2l2、BIRC2、EZR、M6PR 和 SERP1 蛋白水平表达下调。转染 miR-204 的 HTM 细胞在 H(2)O(2)处理后表现出凋亡水平增加、活力降低、氧化蛋白积累增加、ER 应激反应标志物诱导减少以及炎症介质 IL-8 和 IL-11 表达降低。
miR-204 可能在调节 HTM 细胞的多种功能中发挥重要作用，包括凋亡、损伤蛋白积累、ER 应激反应和炎症介质表达。
