microRNA-127抑制葡萄膜黑色素瘤细胞增殖的表观遗传调控研究  

Epigenetic Regulation of MicroRNA-127 in the Inhibition of Uveal Melanoma Cell Proliferation

作　　者：曹琼洁 董菁 李丽 陈晓燕 王教 闫东升 Qiongjie Cao;Jing Dong;Li Li;Xiaoyan Chen;Jiao Wang;Dongsheng Yan(State Key Laboratory of Ophthalmology,Optometry and Vision Science,Eye Hospital of Wenzhou Medical University,Wenzhou 325027,China)

机构地区：[1]温州医科大学附属眼视光医院省部共建眼视光学和视觉科学国家重点实验室,325027

出　　处：《中华眼视光学与视觉科学杂志》2018年第9期546-551,565,共7页Chinese Journal Of Optometry Ophthalmology And Visual Science

基　　金：国家自然科学基金(81272286);国家自然科学青年基金(81301776)。

摘　　要：目的:研究microRNA-127(miR-127)在人葡萄膜黑色素瘤细胞中的表达水平及其对细胞增殖的影响。方法:实验研究。通过实时荧光定量PCR(RT-qPCR)法检测miR-127在葡萄膜黑色素瘤细胞M23和SP6.5与正常葡萄膜黑色素细胞UM95中的表达水平。细胞实验通过阳离子脂质体介导的方法将miR-127和阴性对照(NC)转染入M23和SP6.5中,并应用细胞增殖实验法(MTS法)和流式细胞技术分别检测细胞增殖能力和细胞周期,采用WesternBlot法检测转染miR-127后细胞周期相关蛋白的表达。另外,通过RT-qPCR检测用表观药物5-氮杂-2-脱氧胞苷(5-Aza-dC)和(或)曲古抑菌素A(TSA)处理后的M23和SP6.5细胞内miR-127的表达水平。MiR-127的表达量及细胞实验各参数在2组间比较采用独立样本t检验。结果:miR-127在M23和SP6.5中的表达水平低于UM95(t=72.2、591.5,P<0.001)。MTS结果显示,在M23和SP6.5细胞中,转染miR-127组相对细胞数目较NC组的100%分别减少至(62.3±4.2)%和(65.4±2.3)%,差异具有统计学意义(t=12.7、21.6,P<0.001)。流式细胞技术分析显示,转染miR-127组处于G0/G1期的M23和SP6.5细胞数量比例明显高于NC组(t=-6.7,P=0.003;t=-9.9,P<0.001),同时处于S期的M23和SP6.5细胞数量比例明显低于NC组(t=8.6,P=0.001;t=12.7,P<0.001)。WesternBlot结果表明miR-127可明显降低M23和SP6.5细胞内磷酸化Rb蛋白的表达水平(t=22.2、15.6,P<0.001)。此外,miR-127在M23和SP6.5细胞中的表达可被5-Aza-dC和TSA诱导上调(P<0.05)。结论:miR-127通过抑制细胞周期而抑制人葡萄膜黑色素瘤细胞的增殖,并可被DNA甲基化和组蛋白乙酰化表观遗传机制调控。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 mi

关 键 词：葡萄膜黑色素瘤 microRNA-127 细胞周期 细胞增殖 表观遗传调控 

分 类 号：R739.7[医药卫生—肿瘤]