5-杂氮-2′-脱氧胞苷降低了乳腺癌MDA-MB-231细胞的体外迁移能力

5-Aza-2′-deoxycytidine decreases migration ability of breast cancer MDA-MB-231 cells in vitro

目的探讨5-杂氮-2′-脱氧胞苷(5-Aza-CdR)对高转移性人乳腺癌MDA-MB-231细胞体外迁移能力的作用及可能的机制。方法实验分为对照组与5-Aza-CdR处理组,分别通过Transwell趋化迁移及划痕实验、半定量逆转录PCR(SqRT-PCR)、甲基化特异性PCR(MSP)等方法检测MDA-MB-231细胞的体外迁移能力、乳腺癌转移抑制基因-1(BRMS1)与CXC趋化因子受体-4(CXCR4)基因表达、BRMS1与CXCR4启动子区甲基化状况。结果对照组与处理组细胞穿过Transwell聚碳酸酯滤膜的数量分别为1 290.00±214.64与673.00±44.00,处理组细胞穿膜数量明显减少(P<0.05);24、36、48 h时间点对照组与处理组细胞划痕愈合率分别为20.34±0.69、59.31±0.38、91.77±0.13与20.19±0.67、49.32±0.24、69.47±0.46,其中36 h与48 h时间点处理组愈合率明显降低(P<0.05);对照组与处理组BRMS1的相对于内参GAPDH的灰度值(IDV)分别为0与0.39±0.001,处理组BRMS1 mRNA表达明显上调(P<0.05),5-Aza-CdR使BRMS1启动子区甲基化的CpG岛B完全去甲基化,对照组与处理组CXCR4的相对于内参GAPDH的IDV分别为0.58±0.003与0.58±0.01,两组比较差异无统计学意义(P>0.05),CXCR4启动子区CpG岛1的非甲基化状态亦无明显改变。结论 5-Aza-CdR通过去甲基化机制重新激活肿瘤转移抑制基因BRMS1的表达,从而降低了MDA-MB-231细胞体外迁移能力。

Objective To investigate the migration function of 5-Aza-CdR on high metastatic human breast cancer MDA-MB-231 cell line in vitro and possible mechanisms.Methods MDA-MB-231 cells were treated with 5-Aza-CdR,then cell migration ability in vitro,the mRNA expressions and promotor methylation status of breast cancer metastasis suppressor-1（BRMS1） gene and C-X-C chemokine receptor-4（CXCR4）gene were detected by Transwell chemotactic migration and wound closure assay,semi-quantitative reverse transcription-PCR（SqRT-PCR）,methylation-specific polymerase chain reaction（MSP） respectively.Results 5-Aza-CdR decreased the number of cells passing through polycarbonate filter membrane（1290.00±214.64 versus 673.00±44.00,P0.05）and the rate of wound closure（20.34±0.69,59.31±0.38,91.77±0.13 versus 20.19±0.67,49.32±0.24,69.47±0.46 at 24h,36h,48h time points respetively）at 36h,48h time points（P0.05）;upgraded the BRMS1 mRNA expression（0 versus 0.39±0.001（relative integrated density value,IDV against GAPDH）,P0.05） and demethylated the methylated CpG island B in promotor,while the CXCR4 mRNA expression（0.58±0.003 versus 0.58±0.01,P0.05） and the status of unmethylated CXCR4 CpG island 1 in promotor were not changed significantly.Conclusion 5-Aza-CdR reactivates tumor metastasis suppressor gene BRMS1 and decreases the migration ability of MDA-MB-231 cells in vitro by demethylation mechanism.