MicroRNA-182 Downregulates MITF Expression in Uveal Melanoma Cells

Abstract

Purpose: : MicroRNAs (miRNAs) are endogenous 20-24 nucleotide RNAs which inhibit protein translation through binding to target mRNAs. Recent studies have demonstrated that miRNAs can regulate tumor cell proliferation. The role of miRNAs in uveal melanoma, however, remains largely unknown. In the present study, we investigated the function of (MicroRNA-182) miR-182 in uveal melanoma cells.

Methods: : Northern blotting was performed to detect the expression of miR-182 in uveal melanoma cells and normal melanocytes. The target of miR-182 was predicted by bioinformatics and confirmed by luciferase assay. Uveal melanoma cells were transfected with miR-182. The amount of MITF protein was analyzed by Western blotting to evaluate the effect of miR-182 on MITF. The proliferation of uveal melanoma cells was quantified by MTS assay.

Results: : miR-182 was expressed in normal melanocytes but downregulated in uveal melanoma cells. Three putative miR-182 binding sites were predicted within the 3’ untranslated region (3’ UTR) of human MITF mRNA. Reporter constructs containing the binding sites inserted into the 3’ UTR of the luciferase mRNA conferred a repression of luciferase activity in HEK293 cells transfected with miR-182. miR-182 was found to downregulate the expression of MITF by Western blot analysis. Introduction of miR-182 into uveal melanoma cells led to a significant decrease in cell growth.

Conclusions: : Our results demonstrated that miR-182 may act as a tumor suppressor in uveal melanoma cell proliferation through downregulation of MITF.

Keywords: melanoma • proliferation • gene/expression