KEAP1基因及其突变位点对非小细胞肺癌细胞株作用的实验初步研究

摘要 目的：研究KEAP1基因及KEAP1基因突变位点对肺癌细胞株的作用。方法：通过Western blot 方法，比较携带KEAP1 基因突变的肺癌细胞株（A549,NCI-H460,NCI-H838）与KEAP1 基因野生型的肺癌细胞株（NCI-H292, NCI-H1299, 95D, SPC-A1）之间，NRF2基因与NRF2下游基因HO-1的蛋白表达水平，检测并比较两组细胞的活性氧（ROS）含量；以新发现的非小细胞肺癌（NSCLC）病人的 KEAP1 体细胞突变作为模板构建 KEAP1 突变质粒，对自身存在 KEAP1 突变的肺癌细胞株A549，通过改造的 pMSCV 逆病毒转染体系，分别构建过表达空载，野生型及突变型 KEAP1 的 A549 稳定细胞株，比较过表达不同质粒的细胞间丙二醛（MDA）含量及 NRF2 下游抗氧化等相关基因的表达水平；通过克隆形成实验检测细胞增殖情况。结果：KEAP1基因突变组肺癌细胞株与KEAP1基因无突变的对照组细胞株相比，NRF2和HO-1蛋白表达显著增高，活性氧水平显著降低（P<0.01）；过表达野生型KEAP1 与过表达空载的 A549细胞相比，NRF2 及其下游基因转录水平表达显著下降（P<0.01），蛋白水平表达下降，细胞内丙二醛水平显著增高（P<0.01），克隆形成率显著降低（P<0.01），而过表达突变型 KEAP1 与过表达空载的 A549细胞相比，NRF2 及其下游基因表达、细胞内丙二醛水平、克隆形成率均无显著差异（P>0.05）。结论：KEAP1基因具有抑癌作用，其突变为失活型突变，突变后KEAP1/NRF2通路激活，KEAP1基因突变可能通过改变细胞的氧化应激水平，促进肺癌的发生发展。

The Functional Study of KEAP1 Gene and Its Mutation in NSCLC Cell Line: A Preliminary Experimental Study

ABSTRACT Objective: To investigate the function of mutant KEAP1 and its mutation in lung cancer cells. Methods: Western blot were conducted to determine the expression of Nrf2 and its target gene HO-1 in lung cancer cells with or without KEAP1 mutation. A549-constantly expressing pMSCV, wild type KEAP1 and mutant KEAP1were established by retroviral transfection. The clonogenic assay was performed to explore the effect of wild type Keap1 and mutant Keap1 on the proliferation ability of lung cancer cells. Results: The protein expression of NRF2 downstream gene HO-1 in KEAP1 / NRF2 mutant lung cancer cell lines group were increased compared with the non-mutant lung cancer cell lines group, and the level of reactive oxygen species(ROS) in the mutant group was significantly inhibited compared with the control group(P<0.01). Wide-type KEAP1 overexpression decreased the mRNA and protein expression of NRF2 target genes, increased the MDA level and suppressed proliferation in A549 cells compared with empty vector-transfected A549 cells(P<0.01), while over expression of mutant KEAP1 caused no significant differences(P>0.05). Conclusion: KEAP1 mutation may cause the loss of KEAP1 function, KEAP1mutation may cause tumor genesis and development by changing the oxidative stress of tumor cells.