School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical College, Wenzhou, Zhejiang, China.
Invest Ophthalmol Vis Sci. 2013 Mar 1;54(3):2248-56. doi: 10.1167/iovs.12-10977.
MicroRNA-124a (miR-124a), an abundant microRNA in the central neuron system, has been linked to tumor progression. Here, we investigated the role of miR-124a in uveal melanoma development.
Expression of miR-124a in uveal melanoma cells was examined using real time RT-PCR. The effect of miR-124a on cell proliferation, migration, and invasion was analyzed using MTS assay, flow cytometry, and transwell experiments. The ability of miR-124a to repress tumor growth was tested in vivo. Target genes of miR-124a were first predicted by bioinformatics, confirmed using a luciferase assay, and their expression determined by Western blotting. DNA methylation and histone modification of miR-124a was analyzed by methylation-specific PCR and ChIP assay. Finally, epigenetic drugs were used to alter the expression of miR-124a.
miR-124a expression was downregulated in both uveal melanoma cells and clinical specimens. Transient transfection of miR-124a into uveal melanoma cells inhibited cell growth, migration, and invasion. Moreover, introduction of miR-124a suppressed in vivo growth of tumor. Potential targets of miR-124a were found to include CDK4, CDK6, cyclin D2, and EZH2. Knockdown of EZH2 by siRNA resulted in inhibition of uveal melanoma cell migration and invasion. In addition, miR-124a expression was found to be regulated via epigenetic mechanisms, with its expression restored when cells were treated with a DNA hypomethylating agent, 5-aza-2'-deoxycytidine, and a histone deacetylase inhibitor, trichostatin A.
Our results demonstrated that miR-124a could function as a potent tumor suppressor by regulation of multiple targets, and was epigenetically silenced in the development of uveal melanoma.
miR-124a（miR-124a）是中枢神经系统中丰富的 microRNA，与肿瘤进展有关。在这里，我们研究了 miR-124a 在葡萄膜黑色素瘤发展中的作用。
使用实时 RT-PCR 检查 miR-124a 在葡萄膜黑色素瘤细胞中的表达。使用 MTS 测定、流式细胞术和 Transwell 实验分析 miR-124a 对细胞增殖、迁移和侵袭的影响。体内测试 miR-124a 抑制肿瘤生长的能力。首先通过生物信息学预测 miR-124a 的靶基因，使用荧光素酶测定法确认，并通过 Western blot 测定其表达。通过甲基化特异性 PCR 和 ChIP 测定分析 miR-124a 的 DNA 甲基化和组蛋白修饰。最后，使用表观遗传药物改变 miR-124a 的表达。
miR-124a 在葡萄膜黑色素瘤细胞和临床标本中均下调表达。瞬时转染 miR-124a 可抑制葡萄膜黑色素瘤细胞的生长、迁移和侵袭。此外，引入 miR-124a 抑制肿瘤的体内生长。miR-124a 的潜在靶标包括 CDK4、CDK6、cyclin D2 和 EZH2。用 siRNA 敲低 EZH2 导致葡萄膜黑色素瘤细胞迁移和侵袭受到抑制。此外，发现 miR-124a 的表达受表观遗传机制调控，当细胞用 DNA 去甲基化剂 5-氮杂-2'-脱氧胞苷和组蛋白去乙酰化酶抑制剂曲古抑菌素 A 处理时，其表达得到恢复。
我们的结果表明，miR-124a 可以通过调节多个靶标发挥强大的肿瘤抑制作用，并且在葡萄膜黑色素瘤的发展中被表观遗传沉默。
