MicroRNA-124a Is Epigenetically Regulated and Acts as a Tumor Suppressor by Controlling Multiple Targets in Uveal Melanoma

Abstract

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.