5-杂氮-2’-脱氧胞苷对三阴性乳腺癌细胞肺转移的抑制作用

5-Aza-2＇-deoxycytidine inhibiting metastasis to lungs of triple-negative breast cancer cells

目的探讨5-杂氮-2’-脱氧胞苷（5-Aza—CdR）对三阴性乳腺癌（TNBC）MDA—MB-231细胞实验性肺转移的影响及其机制。方法将MDA—MB-231细胞分对照组与5-Aza—CdR处理组，分别通过半定量逆转录。聚合酶链反应（SqRT．PCR）与甲基化特异性聚合酶链反应（MSP）方法检测乳腺癌转移抑制基因-1（BRMSl）、CXC趋化因子受体-4（CXCR4）基因的mRNA表达与启动子区甲基化状况。然后通过边缘尾静脉将2×10^5个细胞注射到每只BALB／Cnu／nu裸鼠体内（每组5只），5周后，通过荧光定量RT—PCR（FqRT—PCR）检测裸鼠肺组织内目的基因HPRTmRNA丰度来确定癌细胞肺转移程度。结果对照组与处理组细胞的BRMSl相对灰度值（IDV）分别为0与0．390±0．001，处理组BRMSlmRNA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；CXCR4相对IDV分别为0．580±0．003与0．580±0．010，两组之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；接种对照组与处理组细胞的裸鼠肺脏HPRT、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△△Ct0．34，转移抑制率为66％，处理组裸鼠肺脏HPRTmRNA丰度低，转移的癌组织较少。结论5-Aza—CdR通过去甲基化机制重新激活了肿瘤转移抑制基因BRMSI的表达，降低了TNBCMDA—MB-231细胞实验性肺转移的能力。

Objective To investigate the effect of 5-Aza-2＇-deoxycytidine （5-Aza-CdR） on metas- tasis to lungs of triple-negative breast cancer （TNBC） MDA-MB-231 cells and the mechanisms. Methods MDA-MB-231 cells were divided into two groups： control group and 5-Aza-CdR-treated group. The mRNA expression and promotor methylation status of breast cancer metastasis suppressor-1 （ BRMS1 ） and CXC chemokine receptor-4 （CXCR4） in the MDA-MB-231 cells were evaluated by semi-quantitative reverse transcription-polymerase chain reaction （SqRT-PCR） and methylation specific polymerase chain reaction （MSP） respectively. Then 2 x 105 cells were injected into each BALB/C nu/nu mouse （5 mice in each group） through lateral tail veins. Five weeks later, the metastasis of lungs was evaluated through the mRNA abundance of the target gene HPRT in the lungs tissue from the mice detected by fluorescent quantitative RT-PCR （FqRT-PCR）. Results 5-Aza-CdR up-regulated the BRMS1 mRNA expression [0 versus 0. 390 ＋ O. 001 （relative integrated density value, IDV against GAPDH） ,P 〈 0. 05 ] and demethylated the methylated CpG island B in promotor, while the CXCR4 mRNA expression （ 0. 580 ± 0. 003 versus 0. 580 ±0. 010,P 〉0.05 ） and the status of unmethylated CXCR4 CpG island 1 in promotor had no significant change. 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△△Ct =0. 34, and the inhibitory rate of metastasis was 66%. The mRNA abundance of the HPRT was lower and there were fewer metastases in the lungs of 5-Aza-CdR-treated group than in control group. Conclusion 5-Aza-CdR reactivated tumor metastasis suppressor gene BRMS1 and decreased the metastasis to lungs of TNBC MDA-MB-231 cells by demethylation mechanism.