Dong Yong-Qiang, Liang Jiang-Shui, Zhu Shui-Bo, Zhang Xiao-Ming, Ji Tao, Xu Jia-Hang, Yin Gui-Lin
Department of Cardio-Thoracic Surgery, Wuhan General Hospital of Guangzhou Military Command, Wuhan, China.
Asian Pac J Cancer Prev. 2013;14(7):4421-6. doi: 10.7314/apjcp.2013.14.7.4421.
The present study employed 5-aza-2'-deoxycytidine (5-Aza-CdR) to treat non-small cell lung cancer (NSCLC) cell line A549 to investigate the effects on proliferation and expression of the TFPI-2 gene.
Proliferation was assessed by MTT assay after A549 cells were treated with 0, 1, 5, 10 μmol/L 5-Aza-CdR, a specific demethylating agent, for 24 ,48 and 72h. At the last time point cells were also analyzed by flow cytometry (FCM) to identify any change in their cell cycle profiles. Methylation-specific polymerase chain reaction (MSPCR), real time polymerase chain reaction(real-time PCR) and western blotting were carried out to determine TFPI-2 gene methylation status, mRNA expression and protein expression.
MTT assay showed that the growth of A549 cells which were treated with 5-Aza-CdR was significantly suppressed as compared with the control group (0 μmol/L 5-Aza-CdR). After treatment with 0, 1, 5, 10 μmol/L 5-Aza-CdR for 72h, FCM showed their proportion in G0/G1 was 69.7±0.99%, 76.1±0.83%, 83.8±0.35%, 95.5±0.55% respectively (P<0.05), and the proportion in S was 29.8±0.43%, 23.7±0.96%, 15.7±0.75%, 1.73±0.45%, respectively (P<0.05), suggesting 5-Aza-CdR treatment induced G0/G1 phase arrest. MSPCR showed that hypermethylation in the promoter region of TFPI-2 gene was detected in control group (0 μmol/L 5-Aza-CdR), and demethylation appeared after treatment with 1, 5, 10 μmol/L 5-Aza-CdR for 72h. Real-time PCR showed that the expression levels of TFPI-2 gene mRNA were 1±0, 1.49±0.14, 1.86±0.09 and 5.80±0.15 (P<0.05) respectively. Western blotting analysis showed the relative expression levels of TFPI-2 protein were 0.12±0.01, 0.23±0.02, 0.31±0.02, 0.62±0.03 (P<0.05). TFPI-2 protein expression in A549 cells was gradually increased significantly with increase in the 5-Aza-CdR concentration.
TFPI-2 gene promoter methylation results in the loss of TFPI-2 mRNA and protein expression in the non-small cell lung cancer cell line A549, and 5-Aza-CdR treatment could induce the demethylation of TFPI-2 gene promoter and restore TFPI-2 gene expression. These findings provide theoretic evidence for clinical treatment of advanced non-small cell lung cancer with the demethylation agent 5-Aza-CdR. TFPI-2 may be one molecular marker for effective treatment of advanced non-small cell lung cancer with 5-Aza-CdR.
本研究采用5-氮杂-2'-脱氧胞苷（5-Aza-CdR）处理非小细胞肺癌（NSCLC）细胞系A549，以研究其对细胞增殖及组织因子途径抑制物-2（TFPI-2）基因表达的影响。
用0、1、5、10 μmol/L的特异性去甲基化剂5-Aza-CdR处理A549细胞24、48和72小时后，采用MTT法评估细胞增殖情况。在最后一个时间点，还用流式细胞术（FCM）分析细胞，以确定其细胞周期分布的任何变化。采用甲基化特异性聚合酶链反应（MSPCR）、实时聚合酶链反应（real-time PCR）和蛋白质免疫印迹法检测TFPI-2基因的甲基化状态、mRNA表达及蛋白表达。
MTT法显示，与对照组（0 μmol/L 5-Aza-CdR）相比，5-Aza-CdR处理的A549细胞生长受到显著抑制。用0、1、5、10 μmol/L 5-Aza-CdR处理72小时后，FCM显示其在G0/G1期的比例分别为69.7±0.99%、76.1±0.83%、83.8±0.35%、95.5±0.55%（P<0.05），在S期的比例分别为29.8±0.43%、23.7±0.96%、15.7±0.75%、1.73±0.45%（P<0.05），提示5-Aza-CdR处理诱导细胞G0/G1期阻滞。MSPCR显示，对照组（0 μmol/L 5-Aza-CdR）中检测到TFPI-2基因启动子区域的高甲基化，而用1、5、10 μmol/L 5-Aza-CdR处理72小时后出现去甲基化。Real-time PCR显示，TFPI-2基因mRNA表达水平分别为1±0、1.49±0.14、1.86±0.09和5.80±0.15（P<0.05）。蛋白质免疫印迹分析显示，TFPI-2蛋白的相对表达水平分别为0.12±0.01、0.23±0.02、0.31±0.02、0.62±0.03（P<0.05）。A549细胞中TFPI-2蛋白表达随5-Aza-CdR浓度增加而显著逐渐升高。
TFPI-2基因启动子甲基化导致非小细胞肺癌细胞系A549中TFPI-2 mRNA和蛋白表达缺失，5-Aza-CdR处理可诱导TFPI-2基因启动子去甲基化并恢复TFPI-2基因表达。这些发现为临床使用去甲基化剂5-Aza-CdR治疗晚期非小细胞肺癌提供了理论依据。TFPI-2可能是5-Aza-CdR有效治疗晚期非小细胞肺癌的一个分子标志物。
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