自噬对非小细胞肺癌EGFR-TKI耐药的调控作用及其机制研究

发布时间：2018-06-15 18:24

本文选题：EGFR-TKI + LC3-Ⅱ　；参考：《华中科技大学》2016年博士论文

【摘要】：第一部分自噬与非小细胞肺癌EGFR-TKI耐药的关系目的：构建EGFR-TKI耐药细胞株并探索EGFR-TKI耐药与自噬活动的关系。方法：选取4种NSCLC细胞系HCC827, A549, H460和H1975,进行基因检测以验证其EGFR突变状态,并通过CCK8测定其对erlotinib的敏感性。以EGFR-TKI敏感细胞HCC827为基础,通过长期且浓度递增的erlotinib处理以诱导出EGFR-TKI耐药细胞株,并通过CCK8鉴定其对erlotinib的敏感性,命名为HCC827-R。通过Western blotting检测HCC827, A549, H460, H1975和HCC827-R5种细胞的基础自噬水平,并通过GFP-LC3和CQ(chloroquine)进一步验证HCC827和HCC827-R的自噬流水平。通过抑制剂(CQ或3-MA)或siRNA抑制基础自噬观察其对细胞增殖的影响。结果：EGFR基因检测结果为：HCC827(E746-A750 deletion), A549(wild type), H460(wild type), H1975(L858R and T790M),通过CCK8药物敏感性检测发现HCC827对erlotinib敏感,而A549,H460,H1975和HCC827-R对erlotinib抗拒,其中HCC827-R对erlotinib的IC50值大于100gM。综合比较HCC827, A549, H460, H1975和HCC827-R的EGFR-TKI敏感性和基础自噬水平发现,从HCC827到HCC827-R,细胞对erlotinib的药物敏感性逐渐降低,而LC3-Ⅱ水平,即基础自噬水平逐渐增高。通过GFP-LC3和CQ的介入进一步证实,HCC827-R的基础自噬流水平明显高于HCC827。以3-MA或siRNA抑制基础自噬后,HCC827和HCC827-R的增殖未受明显影响。结论：相对于EGFR-TKI敏感细胞,EGFR-TKI耐药细胞拥有更高的基础自噬水平,对EGFR-TKI的敏感性与细胞基础自噬水平之间存在负相关性,基础自噬活动不是细胞增殖的必需因素。第二部分自噬对非小细胞肺癌EGFR-TKI耐药的调控作用目的：进一步探索自噬在NSCLC细胞对EGFR-TKI耐药中的作用以及抑制自噬对EGFR-TKI耐药的影响。方法：通过不同时间不同浓度的erlotinib处理HCC827,A549,H1975和HCC827-R细胞,观察erlotinib对细胞自噬活动的影响。将自噬抑制剂(CQ或3-MA)与erlotinib联合应用于HCC827,A549,H1975和HCC827-R细胞,观察二者的联合作用对自噬活动以及细胞增殖的影响。通过Annexin V和PI双染检测不同浓度的CQ或CQ与erlotinib联用对H1975或HCC827-R凋亡活动的影响,并以Western blotting进一步观察PARP1和caspase 3的变化。在CQ和erlotinib联合作用下,以JC-1染色观察线粒体膜电位的变化,并分别提取胞浆和线粒体蛋白以Western blotting检测胞浆和线粒体中cytochrome c的含量。结果：Erlotinib呈时间依赖性的诱导HCC827,A549,H1975和HCC827-R细胞自噬水平升高,而这一作用可以被自噬抑制剂3-MA或CQ阻断。3-MA或CQ均与erlotinib表现出协同作用,抑制了HCC827, A549, H1975和HCC827-R的细胞增殖,其中3-MA单药对细胞生长无明显的抑制作用,而CQ单药使用时细胞生长和克隆形成均受到显著抑制,提示作用于不同靶点的自噬抑制剂对NSCLC细胞增殖具有不同的影响。CQ与erlotinib联合作用引起了H1975和HCC827-R凋亡水平增高,激活了PARP1与caspase 3,而caspase抑制剂z-VAD fmk逆转了这一联合作用引起的细胞生长抑制。此外,CQ与erlotinib联合应用还引起了线粒体膜电位的丢失和线粒体膜通透性的降低,表现为cytochrome c从线粒体释放入胞浆。结论：自噬抑制剂(3-MA或CQ)抑制了EGFR-TKI引起的自噬反应并进而逆转了NSCLC细胞对EGFR-TKI的耐药,CQ与erlotinib的联合应用通过降低线粒体的膜电位和膜通透性引起NSCLC细胞发生caspase依赖性的凋亡反应。第三部分内质网应激在cQ逆转EGFR-TKI耐药中的作用及机制目的：观察线粒体及内质网活动在CQ逆转EGFR-TKI耐药中的作用。方法：通过GFP-LC3与线粒体示踪剂mitotracker的共定位情况观察线粒体自噬是否参与了CQ与erlotinib的联合作用。在CQ与erlotinib的联合作用下以Western blotting检测内质网应激通路蛋白EIF2α-CHOP的水平,并通过siRNA靶向敲除CHOP观察其对H1975细胞凋亡和细胞增殖的影响。进而,通过JC.1染色和Western blotting检测靶向敲除CHOP后CQ与erlotinib联合作用下线粒体膜电位和膜通透性变化。最后,通过建立H1975移植瘤模型,检测CQ与erlotinib联合作用对H1975移植瘤增殖的影响。结果：在CQ与erlotinib联合作用下,GFP-LC3未能与mitotracker形成荧光共定位,说明线粒体自噬未参与CQ与erlotinib的联合作用。通过Western blotting发现CQ与erlotinib联合作用引起了EIF2α-CHOP通路的活化,说明其引起了内质网应激反应。对CHOP的敲除,逆转了CQ与erlotinib联合作用引起的凋亡反应和细胞生长抑制,并且抑制了线粒体膜电位和膜通透性的变化。在体内试验中,CQ与erlotinib联合作用显著抑制了H1975移植瘤的生长。结论：CQ与erlotinib联合作用通过介导内质网应激反应进而引起线粒体膜电位和通透性的变化,最终引起凋亡,其中CHOP起到了凋亡信号从内质网应激向线粒体凋亡途径传导的中介作用。
[Abstract]:The first part of the relationship between autophagy and EGFR-TKI resistance in non-small cell lung cancer: to construct a EGFR-TKI resistant cell line and explore the relationship between EGFR-TKI resistance and autophagy. Methods: select 4 NSCLC cell lines, HCC827, A549, H460 and H1975, to test their EGFR mutation status and determine their sensitivity to erlotinib by CCK8. On the basis of EGFR-TKI sensitive cell HCC827, the EGFR-TKI resistant cell line was induced by long-term and increasing erlotinib treatment, and the sensitivity to erlotinib was identified by CCK8. It was named HCC827-R. through Western blotting to detect the basic autophagy level of HCC827, A549, H460, and cells. C3 and CQ (chloroquine) further verify the level of autophagic flow in HCC827 and HCC827-R. The effects of autophagy on the basal autophagy of the inhibitor (CQ or 3-MA) or siRNA are observed. Perceptual detection found that HCC827 is sensitive to erlotinib, while A549, H460, H1975 and HCC827-R are resistant to erlotinib. The IC50 values of HCC827-R to erlotinib are greater than 100gM.. Decrease, and LC3- II level, the level of basic autophagy increased gradually. Through the intervention of GFP-LC3 and CQ, the basal autophagy level of HCC827-R was significantly higher than that of HCC827. with 3-MA or siRNA inhibition of the basal autophagy, and the proliferation of HCC827 and HCC827-R was not significantly affected. Conclusion: for EGFR-TKI sensitive cells, EGFR-TKI drug-resistant cells are owned. Higher basic autophagy level, negative correlation between EGFR-TKI sensitivity and cell based autophagy level, basic autophagy is not a necessary factor for cell proliferation. Second the role of autophagy in the regulation of EGFR-TKI resistance in non small cell lung cancer: further explore the role of autophagy in the resistance of NSCLC cells to EGFR-TKI And the effect of inhibition of autophagy on EGFR-TKI resistance. Methods: the effects of erlotinib on the autophagy activity of HCC827, A549, H1975 and HCC827-R cells were observed at different concentrations of erlotinib at different times. The combined effects of the autophagy inhibitor (CQ or 3-MA) and erlotinib were combined with erlotinib to observe the combined effect of the two. The effects on autophagy and cell proliferation. The effects of CQ or CQ on the apoptosis of H1975 or HCC827-R were detected by Annexin V and PI double staining, and the changes of PARP1 and caspase 3 were further observed with Western blotting. The cytoplasm and mitochondrial proteins were extracted with Western blotting to detect the cytochrome C in the cytoplasm and mitochondria. Results: Erlotinib was time dependent induced HCC827, and the autophagy level of A549, H1975 and HCC827-R cells increased, and this effect could be associated with the inhibition of 3-MA or CQ of autophagic inhibitors. The same effect inhibited the cell proliferation of HCC827, A549, H1975 and HCC827-R, in which 3-MA single drug had no obvious inhibitory effect on cell growth, while the cell growth and clone formation of CQ single drug were significantly inhibited, suggesting that the autophagy inhibitors acting at different targets had different effects on the proliferation of NSCLC cells with the combination of.CQ and erlotinib. The effect caused the increase of apoptosis level of H1975 and HCC827-R, activation of PARP1 and caspase 3, and caspase inhibitor z-VAD fmk reverses the cell growth inhibition caused by this combination. In addition, the combination of CQ and erlotinib causes the loss of mitochondrial membrane potential and the decrease of mitochondrial membrane permeability, showing cytochrome c from the mitochondria. Release into the cytoplasm. Conclusion: autophagy inhibitor (3-MA or CQ) inhibits the autophagy induced by EGFR-TKI and then reverses the resistance of NSCLC cells to EGFR-TKI. The combination of CQ and erlotinib can induce caspase dependent apoptosis in NSCLC cells by reducing mitochondrial membrane potential and membrane permeability. Third part of endoplasmic reticulum stress The role and mechanism of cQ in reversing EGFR-TKI resistance: To observe the role of mitochondrial and endoplasmic reticulum activity in reversing EGFR-TKI resistance by CQ. Methods: whether mitochondrial autophagy is involved in the co operation of CQ and erlotinib through the co localization of GFP-LC3 and mitochondrial tracer mitotracker. In combination of CQ and erlotinib The level of endoplasmic reticulum stress pathway protein EIF2 alpha -CHOP was detected by Western blotting, and the effect of siRNA targeting CHOP on the apoptosis and cell proliferation of H1975 cells was observed. Then, the mitochondrial membrane potential and membrane permeability under the joint action of CQ and CQ were detected by JC.1 staining and Western blotting. The effect of the combination of CQ and erlotinib on the proliferation of H1975 xenografts was detected by establishing a H1975 xenograft model. Results: under the combination of CQ and erlotinib, GFP-LC3 failed to form a co localization with mitotracker, indicating that mitochondrial autophagy did not participate in the combination of CQ and erlotinib. The activation caused the activation of the EIF2 alpha -CHOP pathway, indicating that it causes the endoplasmic reticulum stress response. The knockout of CHOP reverses the apoptosis response and cell growth inhibition caused by the combination of CQ and erlotinib, and inhibits the changes in mitochondrial membrane potential and membrane permeability. In vivo tests, the combination of CQ and erlotinib significantly inhibits H197. 5 the growth of transplanted tumor. Conclusion: the combination of CQ and erlotinib can induce the changes of mitochondrial membrane potential and permeability by mediating the endoplasmic reticulum stress response, and eventually induce apoptosis, in which CHOP acts as the intermediary of apoptosis signal transduction from endoplasmic reticulum stress to mitochondrial apoptosis pathway.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2

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