基于增强子表观遗传调控的N-PAHs诱导肺癌肿瘤细胞侵袭转移研究

The outdoor air pollution is confirmed as carcinogenic to humans, and the airborne particulate mixture is most closely associated with increased cancer incidence, especially lung cancer. Previous studies have documented that PM2.5 promoting lung cancer cell metastasis, and NPAHs maybe the important contributor to the effect, because their stronger mutagenic and carcinogenic effects of than PAHs. However, existing research focused mainly on the carcinogenic toxicity of PAHs. It is absent of the experimental evidence about NPAHs promoting lung cancer cell metastasis studies. In the present study, 1）In order "cytoskeleton damage - invasion surrounding cells adhesion of vascular endothelial inflammatory cells - cancer transfer through blood," we will analyse the effects of NPAHs on lung cancer cell metastasis and determined the internal exposure mechanism of the NPAHs. 2） explore mechanisms of NPAHs exposure directly inhibit the “Hippo-YAP” on the early tumor metastasis and caner development. 3）illuminate mechanism of Enhancer regulation epigenetic regulation on the effects of cancer cell migration, invasion and adhesion, provide experimental evidence for study on the health effects of human exposure to NPAHs.

室外空气污染被确定为人类一类致癌物。近期研究提示，PM2.5暴露可诱导人肺癌细胞A549的侵袭转移，二次有机气溶胶（SOA）最主要的组分硝基多环芳烃 （N-PAHs）因具有比其前体PAHs更强致突变和致癌作用而可能成为肺癌肿瘤癌变效应的重要贡献者。为此，本项目拟开展以下工作：（1）按照“癌细胞骨架系统损伤脱离周围癌细胞-裂解细胞基质侵入周围组织-黏附血管内皮炎性细胞-血行转移”的顺序，明确NPAHs对肺癌肿瘤侵袭转移级联效应的影响，解析N-PAHs内暴露机制。（2）阐明N-PAHs直接攻击Hippo-YAP信号通路，诱导肺癌肿瘤侵袭转移级联效应发生的细胞学基础。（3）分析TEAD-YAP转录因子驱动组蛋白修饰活化增强子直接开启癌基因高效转录的表观遗传控机制。旨在为N-PAHs人群暴露健康效应研究提供科学依据。

肺癌是全球最常见的癌症之一，具有极高的死亡率，据世界卫生组织（WHO）估计每年死于肺癌的患者约占全球总癌症死亡病例的1/4。而附着于颗粒物上的硝基多环芳烃（NPAHs），尽管环境中NPAHs的浓度很低，但实验数据已证实NPAHs比PAHs具有更强的直接致突性和致癌性，并把NPAHs归为“可能的人类致癌物”。我们对人支气管上皮细胞BEAS-2B和人肺癌细胞A549，分别采用不同浓度不同浓度1-NP，9-NANT和1-NNAP（1-NP暴露浓度为0, 0.1, 0.3和1μM，9-NANT和1-NNAP暴露浓度为0, 1, 3和10μM）处理24 h，通过划痕实验和Transwell实验分析肿瘤细胞转移和侵袭能力的变化，以及通过细胞黏附内皮层分析试剂盒荧光检测单层培养的HPAECs细胞对肿瘤细胞的黏附能力，确定诱导肺癌肿瘤细胞侵袭-转移的级联效应。其次，通过Western bloting技术检测和分子对接软件计算与LATS的对接情况，确定N-PAHs暴露启动hippo-YAP信号通路发挥其生物毒性。进一步通过考察YAP、p-YAP和TEAD的蛋白表达，采用免疫荧光印记的方法检测胞质内的YAP的分布，明确N-PAHs暴露对胞质内YAP入核情况的影响。同时，检测H3K4me1、H3K4me3和H3K27ac的表达，确定增强子的活化参与了N-PAHs诱导的细胞粘附的调控。采用特异性YAP阻断剂Verteporfin预处理，再进行N-PAHs暴露，检测处理前后E-selectin蛋白表达的情况，从而验证N-PAHs通过激活hippo-YAP信号通路引发肺癌肿瘤细胞侵袭转移级联效应表观遗传机制。最后，我们通过对肺癌模型小鼠进行1-NP鼻腔闭气经口吸入暴露4周，并腹腔注射YAP阻断剂Verteporfin干预处理，在体验证1-NP暴露诱导肺癌肿瘤转移的调控机制。我们研究结果显示，PM2.5附着N-PAHs暴露诱导肺癌肿瘤侵袭转移级联效应的发生，并揭示其可能的表观遗传调控机制。