Abstract
MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play central roles in diverse pathological processes. In this study, we investigated the effect of microRNA-182 (miR-182) on the development of posterior uveal melanomas. Initially, we demonstrated that miR-182 expression was dependent on p53 induction in uveal melanoma cells. Interestingly, transient transfection of miR-182 into cultured uveal melanoma cells led to a significant decrease in cell growth, migration, and invasiveness. Cells transfected with miR-182 demonstrated cell cycle G1 arrest and increased apoptotic activity. Using bioinformatics, we identified three potential targets of miR-182, namely MITF, BCL2 and cyclin D2. miR-182 was shown to have activity on mRNA expression by targeting the 3′ untranslated region of MITF, BCL2 and cyclin D2. Subsequent Western blot analysis confirmed the downregulation of MITF, BCL2 and cyclin D2 protein expression. The expression of oncogene c-Met and its downstream Akt and ERK1/2 pathways was also downregulated by miR-182. Concordant with the findings that miR-182 was decreased in uveal melanoma tissue samples, overexpression of miR-182 also suppressed the in vivo growth of uveal melanoma cells. Our results demonstrated that miR-182, a p53 dependent miRNA, suppressed the expression of MITF, BCL2, cyclin D2 and functioned as a potent tumor suppressor in uveal melanoma cells.
Highlights
Uveal melanoma is a tumor arising out of pigmented cells of the eye including the iris, ciliary body, or choroid [1]
Western blot analysis showed that microphthalmiaassociated transcription factor (MITF) was dramatically reduced when cells were transfected with miR-182 (Fig. 6A). c-Met, which is a target of MITF, was decreased in M23 and SP6.5 cells transfected with miR-182 in comparison to either mock or a negative control transfected cells (Fig. 6B). c-Met has been shown to activate diverse intracellular signaling pathways including Akt and ERK1/2 [25]
Since c-Met is a direct target of MITF, we investigated the effect of c-Met on uveal melanoma cells. c-Met specific siRNA was used to decrease the expression of c-Met in both M23 and SP6.5 cells (Fig. 7A)
Summary
Uveal melanoma is a tumor arising out of pigmented cells of the eye including the iris, ciliary body, or choroid [1]. Posterior uveal melanomas frequently harbor GNAQ mutations, but rarely BRAF mutations [4,5] These tumors behave aggressively and frequently present with hematogenous metastases to the liver early in the course of disease progression [1,6]. Recent studies have confirmed that miRNAs may have a role in the regulation of metastatic melanoma with alterations in the levels of the c-Met and MITF gene [10,14]. Studies revealed that miR-34a is a proapoptotic transcriptional target of the p53 tumor suppressor gene, with consequent effects on a variety of tumor types [14,18,19,20]. MiR182 was found to function as a component of the p53 network and a tumor suppressor in posterior uveal melanoma cells
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