肺腺癌细胞DR4基因启动子甲基化与TRAIL敏感性的相关性研究

目的 探讨DR4基因启动子甲基化水平与肺腺癌细胞株对肿瘤坏死因子相关凋亡诱导配体（TRAIL）敏感性之间的相关性。方法 选取未经5-氮杂-2'-脱氧胞苷（5-Aza-CdR）处理和10 μmol/L 5-Aza-CdR处理3 d后的肺腺癌细胞A549、LTEP-α-2，采用CCK-8法检测细胞增殖抑制率，倒置显微镜下观察细胞形态学改变，流式细胞仪检测细胞凋亡率；采用RT-PCR法、免疫印迹法和甲基化特异性PCR（MSP）法分别检测肺腺癌细胞株（A549、LTEP-α-2）DR4基因mRNA、蛋白表达和启动子区甲基化状态。结果 肺腺癌细胞（A549、LTEP-α-2）对低浓度TRAIL高度耐受，提高TRAIL浓度（15.625、31.25、62.5、125、250、500 μg／ml）作用细胞24、48 h后，细胞生长受到不同程度抑制，且呈剂量和时间依赖性；经5-Aza-CdR处理后，TRAIL对肺腺癌细胞的增殖抑制作用均较处理前显著增强（P＜0.05），倒置显微镜下细胞形态表现出变圆、皱缩甚至脱落等特征。应用5-Aza-CdR处理后，125 μg／ml TRAIL诱导肺腺癌细胞的凋亡率较处理前明显升高（P＜0.05）。A549、LTEP-α-2细胞存在DR4 mRNA及蛋白的低表达，其基因启动子处于甲基化状态；经5-Aza-CdR处理后，肺腺癌细胞中DR4 mRNA及蛋白的表达均显著升高（P＜0.05），其基因启动子处于非甲基化状态。结论 5-Aza-CdR可以逆转DR4基因启动子甲基化状态，上调基因表达，增强TRAIL诱导肺腺癌细胞凋亡的能力，从而逆转TRAIL耐药。5-Aza-CdR联合TRAIL可能是治疗肺腺癌的一种新策略。

Relationship between DR4 gene promoter methylation and TRAIL resistant in lung adenocarcinoma

Objective To investigate the relationship between DR4 gene promoter methylation and tumor necrosis factor related apotosis inducing ligand（TRAIL） resistant in lung adenocarcinoma. Methods Lung adenocarcinoma cells A549, LTEP-α-2 untreated by 5-aza-2'-deoxycytidine（5-Az-CdR） and treated by 5-Aza-CdR for 3 days were selected. Cell counting kit-8（CCK-8）assay was used to determine the inhibitory rate of cell proliferation，and the cell morphological was observed by inverted microscope. The apoptosis of cells was detected by flow cytometry. The protein and mRNA expression levels of DR4 gene in lung adenocarcinoma cell lines（A549， LTEP-α-2） were detected by Western blotting and RT-PCR methods. The methylation of DR4 gene promoter region was examined by methylation-specific PCR（MSP）. Results Lung adenocarcinoma cells（A549， LTEP-alpha-2） were highly resistance to TRAIL at the low concentration. After increasing the concentration of TRAIL（15.625, 31.25, 62.5, 125, 250, 500 μg/ml）， cell growth was inhibited and existed in a dose and time dependent after 24 and 48 hours. After cells were treated by 5-Aza-CdR， the inhibitory effects of lung adenocarcinoma cell treated by TRAIL was significantly higher than before treatment（P＜0.05）.The morphology of the cells was round， shrinkage and even shedding under the microscope. After cells were treated by 125 μg／ml 5-Aza-CdR， the apoptosis rate of lung adenocarcinoma cells induced by TRAIL was significantly much more higher than before treatment（P＜0.05）. Expressions of DR4 mRNA and protein in A549， LTEP-α-2 cells were low， and DR4 gene promoter was methylation state. After cells were treated by 5-Aza-CdR， the expressions of DR4 mRNA and protein were more increased significantly than before treatment（P＜0.05）， and DR4 gene promoter was unmethylated. Conclusion 5-Aza-CdR can reverse DR4 gene promoter methylation state， regulating the level of gene expression， increasing TRAIL induced apoptosis in lung cancer cells， furthermore， reversing TRAIL resistance. Therefore， 5-Aza-CdR combined with TRAIL may be a new strategy for the treatment of lung adenocarcinoma.