PM2.5致ErbB-2/ErbB-4基因低甲基化在肺癌发生过程中的作用及其机制研究

Lung cancer ranks the first in cancer incidence and mortality in our country. Fine particulate matter has been classified by the International Agency for research on cancer as humans carcinogens that can induce the lung cancer. Our previously study found that PM2.5 can lead to hypomethylation of ErbB gene that closely related to lung cancer. However, the mechanism is not clear. We investigate the function of hypomethylation of ErbB and its mechanism in the progress of lung cancer induced by PM2.5 through cellular models, animal models and samples of lung cancer in non-smokers. In vitro, we study the PM2.5-induced toxicities, such as oxidative damage and DNA damage. We also investigate the abnormal cell proliferation, cell malignant transformation and the capacity of migration infiltration. The regulation of ErbB methylation to downstream signaling pathways of Ras and PI3K/Akt are analyzed through the reverse-verified of inhibitors of methylation and gene silencing technology. We observe the capcity of evil cells into cancer through animal experiments and the carcinogenic effects of subchronic exposure of PM2.5. The regulation of ErbB methylation to the signaling pathways are explored. We evaluate ErbB methylation levels, mRNA and protein levels by adapting lung cancer samples and carcinoma tissues of non-smokers. Our research will provide scientific evidence for air pollution control and lung cancer prevention in China.

肺癌居我国肿瘤发病率和死亡率之首，大气颗粒物可诱发肺癌，已被国际癌症研究机构列为人类确定致癌物。课题组前期研究发现，PM2.5可导致与肺癌密切相关的ErbB基因低甲基化，但其作用机制尚不清楚。本课题采用细胞模型、动物模型和非吸烟人群肺癌样本系统研究PM2.5致ErbB基因低甲基化在肺癌发生过程中的作用及分子机制。体外条件下研究PM2.5致细胞氧化损伤和DNA损伤等毒性表型及细胞异常增殖、细胞恶性转化和迁移浸润能力，分析ErbB基因甲基化对下游信号通路Ras/Raf/MAPK和PI3K/Akt的调控作用，通过甲基化抑制剂和基因沉默技术进行反向验证；动物实验观察恶转细胞成瘤能力及PM2.5亚慢性暴露的致癌作用，分析ErbB基因甲基化对肺癌信号通路的调控作用；采用非吸烟人群肺癌组织和癌旁组织进行ErbB基因甲基化水平、mRNA水平和蛋白水平验证。研究将为我国大气污染治理和肺癌防治提供科学依据。

课题组前期研究发现，PM2.5可导致与肺癌密切相关的基因甲基化水平改变，但其作用机制尚不清楚。DNA甲基化芯片技术检测结果表明，PM2.5引起的单核苷酸水平CpG甲基化位点差异为1963个，与非小细胞肺癌相关通路、AMPK信号通路、ErbB信号通路、Ras信号通路等密切有关。对关键基因进行验证发现，PM2.5可显著上调CXCL1、ACTN4、MARK2等，并下调PSMA3和PSMD1。体外研究发现，PM2.5可降低BEAS-2B细胞存活率，增加LDH酶活性和ROS水平，导致氧化损伤、DNA损伤和多核形成，激活HER2/ErbB2/Ras信号通路和原癌基因c-Mcy。PM2.5染毒BEAS-2B细胞30代后，ERBB4甲基化水平降低；细胞多核发生率、DNA损伤程度明显高于对照组，表明PM2.5染毒长期暴露可导致细胞基因组不稳定，且上皮标志物E-Cadherin表达降低与间质标志物Vimentin、N-Cadherin表达升高，提示其促进上皮-间质转化作用，但细胞划痕实验和裸鼠成瘤实验为阴性。体内研究表明：PM2.5亚慢性染毒大鼠1个月后，血清中癌胚抗原CEA的含量升高。肺部PET/CT断层扫描结果分析发现，PM2.5暴露停止后的6、12、18个月时点，肺部18F-FDG的SUV值高于对照组。肺组织ERbB2，ERbB4，MEK，Erk1/2蛋白表达水平升高，且具有剂量依赖趋势。病理学检测发现，PM2.5染毒组出现炎细胞浸润，支气管壁增生，肺泡壁增厚。肺泡灌洗液总蛋白、LDH、炎症因子IL-6，IL-1β，TNF-α的含量均显著升高。短期PM2.5暴露未引起羟脯氨酸含量变化；在暴露停止后的6、12、18个月时点，染毒组大鼠肺部胶原含量和羟脯氨酸含量增多，肺组织RelA/p65表达水平升高，引起上皮-间质转化。上述研究成果可为大气污染相关呼吸系统疾病防治提供科学依据。