Objective: We investigated the expression and regulation of microRNA-127 (miR-127) in uveal melanoma cells. We also investigated the effect of epigenetically upregulating miR-127 by altering DNA methylation and histone acetylation. Methods: In this experimental study, we performed real-time reverse transcriptase
quantitative polymerase chain reaction (RT-qPCR) to detect the expression level of miR-127 in both uveal melanoma cells (M23 and SP6.5) and uveal melanocytes (UM95). Lipofectamine RNAiMAX reagent was used to transfect M23 and SP6.5 with either miR-127 or a negative control (NC). The proliferation of uveal melanoma cells was quantified by 3-[4, 5-dimethyl-thiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazo-lium, inner salt (MTS) cell proliferation assay. Cell cycle was examined by flow cytometry. The expression of cell cycle-related proteins was analyzed by Western Blot. In addition,RT-qPCR was performed to detect the expression level of miR-127 in M23 and SP6.5 cells after treatment with 5-aza-2-deoxycytidine (5-Aza-dC) and trichostatin A (TSA). These agents modify gene expression through DNA methylation and histone acetylation, respectively. Data were analyzed using independent t-tests. results: miR-127 was dramatically downregulated in M23 and SP6.5 cells as compared to UM95 (t=72.2, 591.5, P<0.001). The MTS assay showed that the relative number of cells transfected with miR-127 [(62.3±4.2)% and (65.4±2.3)%] was significantly lower than transfected with NC (t=12.7,21.6, P<0.001). Flow cytometry showed that the percentage of M23 and SP6.5 cells transfected with miR-127 in G0/G1 phase was significantly higher than for the NC group (t=-6.7, P=0.003; t=-9.9,P<0.001), and the percentage of M23 and SP6.5 cells in the S phase was significantly lower than for the NC group (t=8.6, P=0.001; t=12.7, P<0.001). Furthermore, miR-127 downregulated the level of phosphorylated retinoblastoma protein in uveal melanoma cells. In addition, miR-127 was upregulated after treatment with 5-Aza-dC and/or TSA (P<0.05). conclusions: Our results demonstrated that miR-127 can suppress uveal melanoma cell proliferation by inhibition of cell cycle. Furthermore miR-127 expression was modulated by epigenetic regulation including DNA methylation and histone acetylation.
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