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目的研究肺癌细胞和人正常支气管细胞(human bronchial epithelial cell,HBE)中NF-κB2的表达情况及其启动子区CpG岛的甲基化状态。方法采用实时荧光定量PCR(Real-time PCR)方法检测肺癌细胞和正常支气管细胞NF-κB2基因mRNA的表达情况;采用Western blot技术检测NF-κB2前体蛋白p100的表达情况;采用重亚硫酸盐测序(Bisulfite Sequencing PCR,BSP)技术检测NF-κB2启动子区CpG岛的甲基化状态。结果肺腺癌细胞株A549、肺鳞癌细胞株SK-MES-1和小细胞肺癌细胞株NCI-H446的NF-κB2 mRNA的表达分别是HBE细胞株的6.42±0.91倍(P<0.05,t=5.828)、2.78±0.52倍(P<0.05,t=3.219)、2.79±0.33倍(P<0.05,t=4.611);3种肺癌细胞中p100蛋白较HBE细胞均上调表达;4种细胞株中所扩增片段的CpG位点均呈非甲基化状态。结论肺癌细胞及HBE细胞中NF-κB2启动子区域的CpG位点处于非甲基化状态,说明NF-κB2基因表达情况与甲基化状态无直接关系,其启动子区的甲基化修饰未直接参与该基因的表达调控,可能存在其他相关的调控机制,有待进一步的探索。
Objective To investigate the expression of NF-κB in lung cancer cells and human normal human bronchial epithelial cells (HBE) and the methylation status of CpG island in promoter region. Methods Real-time PCR was used to detect the mRNA expression of NF-κB2 in lung cancer cells and normal bronchial cells. The expression of NF-κB p100 protein was detected by Western blot. Bisulfite Sequencing PCR (BSP) was used to detect the methylation status of CpG island in NF-κB promoter region. Results The expressions of NF-κB2 mRNA in lung adenocarcinoma cell line A549, lung squamous carcinoma cell line SK-MES-1 and small cell lung cancer cell line NCI-H446 were 6.42 ± 0.91 times (P <0.05, t = 5.828), 2.78 ± 0.52 (P <0.05, t = 3.219), 2.79 ± 0.33 (P <0.05, t = 4.611) .The levels of p100 in all three kinds of lung cancer cells were up- The CpG sites of the amplified fragments were all non-methylated. Conclusion The CpG locus of NF-κB2 promoter region in lung cancer cells and HBE cells is unmethylated, indicating that the expression of NF-κB2 gene is not directly related to the methylation status, and the methylation of its promoter region Directly involved in the expression of the gene regulation, there may be other related regulatory mechanisms, pending further exploration.