Original Article
Epigenetic Regulation of MicroRNA-127 in the Inhibition of Uveal Melanoma Cell Proliferation
Qiongjie Cao, Jing Dong, Li Li, Xiaoyan Chen, Jiao Wang, Dongsheng Yan
Published 2018-09-25
Cite as Chin J Optom Ophthalmol Vis Sci, 2018, 20(9): 546-551,565. DOI: 10.3760/cma.j.issn.1674-845X.2018.09.007
Abstract
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.
Key words:
uveal melanoma cell; microRNA-127; cell cycle; proliferation; epigenetic regulation
Contributor Information
Qiongjie Cao
State Key Laboratory of Ophthalmology, Optometry and Vision Science, Eye Hospital of Wenzhou Medical University, Wenzhou 325027, China
Jing Dong
Li Li
Xiaoyan Chen
Jiao Wang
Dongsheng Yan
