5-氮杂-2′-脱氧胞苷对肺癌WIF1、RASSF2A基因表达及细胞凋亡的影响

Effect of 5-Aza-CdR on Expression of WIF1 and RASSF2A Gene and Apoptosis in Lung Cancer

目的观察在肺腺癌SPC-A-1细胞中5-氮杂-2′-脱氧胞苷(5-Aza-CdR)对WIF1、RASSF2A基因甲基化状态改变的影响,探讨5-Aza-CdR对肺腺癌SPC-A-1细胞凋亡的影响及其可能的抗癌机制。方法将体外培养的人肺腺癌SPC-A-1细胞株分为4组,A组为未加药组(对照组),B、C、D加药组分别加入3、6、12μmol/L的5-Aza-CdR。采用甲基化特异性PCR(MSP)方法检测4组细胞WIF1、RASSF2A基因甲基化状态的差异,RT-PCR法检测4组细胞WIF1、RASSF2A基因mRNA表达水平差异,Western blot检测4组细胞WIF1、RASSF2A蛋白表达水平差异,流式细胞学技术检测各组细胞周期及细胞凋亡率的差异。结果①A组细胞检测出WIF1、RASSF2A基因为甲基化状态,B、C、D组均检测出WIF1、RASSF2A为去甲基化状态。②肺腺癌SPC-A-1细胞WIF1、RASSF2A基因mRNA及WIF1、RASSF2A蛋白表达水平低下,经5-Aza-CdR处理后,其表达水平明显增强(均P<0.05)。③5-Aza-CdR处理后的细胞凋亡率较未加药组均明显提高,差异有统计学意义(均P<0.05)。④5-Aza-CdR处理后的D组细胞WIF1、RASSF2A蛋白表达水平与细胞凋亡率呈正相关(均P<0.05)。结论肺癌细胞WIF1、RASSF2A基因呈甲基化状态,5-Aza-CdR具有明显去甲基化作用,可使WIF1、RASSF2A基因重新激活表达,进而促进肺癌细胞凋亡及抑制癌细胞增殖,发挥抗癌作用。

Objective To observe the effects of 5-aza-2′-deoxycytidine(5-Aza-CdR)on the methylation status changes of WIF1 and RASSF2 A in lung adenocarcinoma cells SPC-A-1,and to investigate the effects of 5-Aza-CdR on apoptosis of lung adenocarcinoma cell line SPC-A-1 and its possible mechanism.Methods The cultured human lung adenocarcinoma cells SPC-A-1 were divided into four groups:group A(no dosing group)and group B,C,D(treated with 3,6,12 μmol/L 5-Aza-CdR).Differences in WIF1 or RASSF2 A gene methylation status among the four groups were detected by methylation-specific PCR(MSP).Differences in mRNA expression and protein expression of WIF1 or RASSF2 A gene among the different concentration groups were detected by RT-PCR and Western blotting,respectively.Furthermore,differences of the cell cycle and apoptosis rate of four groups were detected by flow cytometry.Results ① WIF1 or RASSF2 A gene of specimens in group A exhibited methylation status,while the group B,C and D exhibited demethylation status.②The mRNA and protein expression levels of WIF1 or RASSF2 A in lung cancer cells SPC-A-1 were very low before treatment.After 5-Aza-CdR treatment,the mRNA and protein expression levels of WIF1 or RASSF2 A were significantly enhanced(P<0.05).③Apoptosis rate of dosing group significantly increased compared with no dosing group,the difference was statistically significant(P<0.05).④The protein expression levels of WIF1 and RASSF2 A in group D were positively correlated with the apoptosis rate(P<0.05).Conclusion WIF1 and RASSF2 A gene in lung cancer have defective expression due to methylation.5-Aza-CdR has a obvious demethylation effect.WIF1 and RASSF2 A gene expression can be reactivated by 5-Aza-CdR.5-Aza-CdR promotes apoptosis and inhibits cell proliferation in lung cancer,and thus plays a role in cancer treatments.