5-杂氮-2′-脱氧胞苷对实验性乳腺癌肺转移的影响

Effect of 5-Aza-2′-deoxycytidine on experimental lung metastasis of breast cance

目的探讨5-杂氮-2′-脱氧胞苷(5-Aza-CdR)对乳腺癌MDA-MB-231细胞实验性肺转移的影响及可能的机制。方法将MDA-MB-231细胞分为对照组与5-Aza-CdR处理组,通过半定量RT-PCR(SqRT-PCR)与甲基化特异性PCR(MSP)方法检测两组细胞的乳腺癌转移抑制基因-1(BRMS1),CXC趋化因子受体-4(CXCR4)基因的mRNA表达及启动子区甲基化的状况。分别将两组细胞通过边缘尾静脉注射至BALB/c nu/nu裸鼠体内(每组5只)。5周后,用荧光定量RT-PCR(FqRT-PCR)检测裸鼠肺组织内的目的基因HPRT及内参GAPDH的mRNA丰度。结果对照组和处理组细胞的BRMS1相对灰度值(IDV)分别为0与0.39±0.001,处理组BRMS1 mRNA表达较对照组明显上调(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;CXCR4相对IDV分别为0.58±0.003与0.58±0.01,两组间差异无统计学意义(P>0.05),CXCR4启动子区CpG岛1的非甲基化状态亦无明显改变。对照组与处理组细胞的裸鼠肺脏HPRT和GAPDH的Ct值分别为:24.75±1.55,16.19±0.69与27.61±1.67,17.48±0.96,2-ΔΔCt=0.34,处理组裸鼠肺脏HPRTmRNA丰度明显低于对照组,肺组织内转移癌较少。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细胞实验性肺转移能力。

Objective To investigate the effect of 5-Aza-CdR on experimental lung metastasis of breast cancer and the possible mechanisms.Methods MDA-MB-231 cells were divided into control group and 5-Aza-CdR-treated group.The mRNA expressions and promotor methylation status of BRMS1 and CXCR4 of the MDA-MB-231 cells were evaluated by SqRT-PCR and MSP respectively.Then,the MDA-MB-231 cells of control group and 5-Aza-CdR-treated group were injected into BALB/c nude mice through lateral tail veins,respectively.five weeks later,the mRNA abundance of the target gene HPRT and internal control gene GAPDH of the lung tissues from the mice were evaluated by FqRT-PCR.Results 5-Aza-CdR upgraded the BRMS1mRNA expression significantly than that in control group（0 versus 0.39±0.001,P〈0.05） and demethylated the methylated CpG island B in promotor,while the CXCR4mRNA expression（0.58±0.003 versus 0.58±0.01,P〉0.05） and the status of unmethylated CXCR4 CpG island 1 in promotor were not changed significantly.The Ct values of HPRT and GAPDH in control and 5-Aza-CdR-treated groups were 24.75±1.55,16.19±0.69 versus 27.61±1.67,17.48±0.96 respectively,2-ΔΔCt=0.34.The mRNA abundance of the HPRT was lower and there were fewer metastases in the lungs of 5-Aza-CdR-treated group compared with control group.Conclusions 5-Aza-CdR can activate tumor metastasis suppressor gene BRMS1 by demethylation mechanism,and thus,decreased the ability of breast cancer MDA-MB-231 cells for experimental metastasis to lungs.