5-Aza-CdR对非小细胞肺癌细胞株A549细胞TFPI-2基因甲基化水平及表达的影响

Effects of 5-aza-2-deoxycytidine on methylation and expression of tissue factor pathway inhibitor-2 gene in non-small cell lung cancer A549 cells

目的:探讨甲基化酶抑制剂5-氮杂-2′-脱氧胞苷(5-aza-2-deoxycytidine,5-Aza-CdR)对非小细胞肺癌(NSCLC)细胞株A549中抑癌基因组织因子途径抑制物2(TFPI-2)基因甲基化水平及基因表达的影响。方法:用不同浓度的5-Aza-CdR[(1、5、10)×10-6mol/L]处理肺癌细胞株A549细胞,不加药物(0 mol/L)作为对照组。药物处理72 h后,甲基化特异性PCR(MSP)检测A549细胞TFPI-2基因的甲基化状态;Real-time PCR技术检测A549细胞TFPI-2基因mRNA的表达;Western blot检测A549细胞TFPI-2蛋白的表达。结果:MSP检测到对照组A549细胞TFPI-2基因呈高甲基化状态,5-Aza-CdR处理异常甲基化得到逆转。Real-time PCR检测显示(0、1、5、10)×10-6mol/L的5-Aza-CdR处理A549细胞72 h后,相对mRNA表达水平分别为1±0、1.49±0.14、1.86±0.09、5.80±0.15(P<0.05),TFPI-2基因mRNA表达呈明显的上升趋势,且随着药物浓度增加而增加。Western blot分析结果显示,对照组与(1、5、10)×10-6mol/L的5-Aza-CdR组的TFPI-2蛋白表达相对水平分别为0.12±0.01、0.23±0.02、0.31±0.02、0.62±0.03(P<0.05)。结论:5-Aza-CdR能成功逆转肺癌A549细胞中TFPI-2基因的高甲基化状态,并恢复TFPI-2基因mRNA及蛋白的表达。

Objective To investigate the effects of 5-aza-2-deoxycytidine （5-Aza-CdR） on methylation and expression of tissue factor pathway inhibitor-2 （TFPI-2） gene in non-small cell lung cancer A549 cells. Methods Lung cancer cell line A549 cells were treated with 5-Aza-CdR in different concentrations [ （1,5,10） × 10^-6 tool/L], without drugs as the control group. After 5-Aza-CdR treatment for 72 h, methylation status of TFPI-2 gene of A549 cells were detected by methylation specific PCR （MSP）; the expression of TFPI-2 gene mRNA were detected by Real-time PCR; the expression of TFPI-2 protein were detected by Western blot. Results TFPI-2 gene of A549 cells was in high methylation status in control group, abnormal methylation was effectively reversed by 5-Aza-CdR treatment. After （0, 1, 5, 10） ×10^-6 mol/L 5-Aza-CdR treatment for 72 h, relative mRNA expression level were 1 ± O, 1.49 ± 0.14, 1.86 ± 0.09, 5.80 ± 0.15 respectively（P 〈 0.05）, TFPI-2 gene mRNA expression obviously increased with the 5-Aza-CdR concentration increasing. The relative expression levels of TFPI-2 protein in （1, 5, 10） × 10^-6 mol/L 5-Aza-CdR group were, 0.23 ± 0.02, 0.31 ± 0.02, 0.62 ± 0.03 respectively,significantly higher than the control group （0.12 ±0.01） （P 〈 0.05）. Conclusion 5-Aza-CdR treatment for lung cancer A549 cells can successfully reverse high methylation status of TFPI-2 gene, and restore the expression of TFPI-2 gene mRNA and protein.