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。
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 KEAP1 were 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 m RNA 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).Conclusions:KEAP1 mutation may cause the loss of KEAP1 function,KEAP1 mutation may cause tumor genesis and development by changing the oxidative stress of tumor cells.
作者
李燕
龚美玲
叶小萍
张琳琳
郑翠侠
LI Yan;GONG Mei-ling;YE Xiao-ping;ZHANG Lin-lin;ZHENG Cui-xia(Department of Respiration,Shanghai Ninth People's Hospital,Shanghai Jiaotong University School of Medicine,Shanghai,200011,China;Yangpu Hospital,Tongji University,Shanghai,200082,China;The Core Laboratory in Medical Center of Clinical Research,Shanghai Ninth People's Hospital,Shanghai Jiaotong University School of Medicine,Shanghai,200011,China;Shanghai Traditional Chinese Medicine Integrated Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai,200082%China)
出处
《现代生物医学进展》
CAS
2021年第7期1201-1207,共7页
Progress in Modern Biomedicine
基金
国家自然科学基金项目(81472177
81772460)。
