M2胆碱受体调控非小细胞肺癌的恶性表型及其信号途径的研究

发布时间：2018-05-27 21:50

本文选题：M2 + mAChR　；参考：《上海交通大学》2015年博士论文

【摘要】：众所周知,胆碱受体(AChR)在介导递质乙酰胆碱(acetylcholine,ACh)神经信号传递过程中起着关键作用。越来越多研究显示非神经源性ACh作为一种局部信号分子或细胞因子以自分泌或旁分泌方式参与多种细胞的生物学功能调控,微环境中ACh与肿瘤细胞的恶性表型(包括过度增殖、转移、耐药及肿瘤干细胞)调控备受学术界关注。ACh作为配体既可作用于门控通道型烟碱型胆碱受体(nAChR),又可作用于G-蛋白耦联型M1组(M1、M3、M5亚型)和M2组(M2、M4亚型)毒蕈碱型胆碱受体(mAChR)。相同的信号分子ACh作用于不同类型受体及其亚型,其受体水平和受体后信号通路水平上生物学功能互作和整合是ACh调控肿瘤细胞中极为重要的科学问题。由于吸烟是肺癌高危因素,烟草主要成分尼古丁(烟碱)激活nAChR调控肺癌细胞的研究较为深入,M1组mAChR对肺癌细胞增殖和上皮间质转化(epithelial to mesenchymal transition,EMT)体外作用也有研究,而M2组mAChR对肺癌恶性表型的体内外调控作用迄今尚未见报道。肺癌靶向治疗业已获得重大进展,但获得性耐药仍然是治疗难题,肿瘤干细胞学说认为肺癌耐药与肿瘤干细胞密切相关。胆碱受体在分子靶向药物的耐药和肺癌干细胞的干性维持的作用值得进一步探讨。为此,本课题重点围绕M2型mAChR在体内和体外水平上对非小细胞肺癌(NSCLC)增殖、转移以及耐药和肺癌干细胞的调控作用及其信号途径开展研究。首先,采用CCK-8、RTCA及Western blot等方法检测NSCLC细胞A549、PC9中非神经源性胆碱能系统的存在。通过mAChR各亚型选择性拮抗剂对NSCLC细胞的增殖和调控MAPK和AKT信号通路作用,揭示M2 mAChR起到明显正向调控作用。M2 mAChR shRNA方法进一步证实其作用和信号途径。荷瘤实验表明,M2 mAChR拮抗剂美索曲明(Methoctramine,MT)和M2R-shRNA均显著延缓A549细胞的体内生长。其次,采用Western blot、transwell、免疫荧光等方法考察了MT及M2 mAChR shRNA抑制NSCLC细胞A549和PC9发生EMT、侵袭和迁移过程。采用MT明显逆转NSCLC细胞A549体内发生EMT并减少转移灶的形成。MT及M2 mAChR siRNA可显著抑制肿瘤转移相关的NFκB信号通路,及其下游转录因子snail和zeb1表达。再次,建立耐EGFR靶向药物吉非替尼(Gefitinib,Ge)的NSCLC细胞株PC9-GR和干性标志CD221阳性的NSCLC干细胞CD221+-SPC-A1,M2 mAChR拮抗剂逆转PC9-GR细胞EMT,CD221+-SPC-A1细胞中M2 mAChR表达明显升高(p0.01),且伴有EMT的变化(相较于CD221-SPC-A1细胞)。本课题首次揭示了M2 mAChR在体内外均可对NSCLC增殖、EMT、迁移、侵袭起到的正向调控作用,并阐明了增殖作用由PI3K/AKT、MEK/ERK信号通路介导,EMT、迁移和侵袭过程由NFκB/snail/zeb1信号通路介导;M2 mAChR可能在NSCLC耐药产生和干细胞的干性维持起到一定调控作用。上述研究成果为非神经源性胆碱能系统对肺癌恶性表型的调控机制阐明和研发M2亚型高选择性拮抗药潜在应用提供理论依据。
[Abstract]:It is well known that acetylcholine receptor (AChR) plays a key role in the transmission of acetylcholine (acetylcholine). More and more studies have shown that non-neurogenic ACh, as a local signal molecule or cytokine, participates in the regulation of biological functions of many kinds of cells in an autocrine or paracrine manner. ACh is associated with malignant phenotypes (including hyperproliferation) of tumor cells in microenvironment. The regulation of metastasis, drug resistance and tumor stem cell) has attracted the attention of academia. Ach as ligand can not only act on the gated channel type nicotinic choline receptor (nAChRN), but also on the G protein coupled type M1 group (M 1, M 3, M 3, M 5 subtype) and M2 group, the M 2N M 4 subtype of muscarinic receptor, muscarinic receptor mAChRN. The same signal molecule ACh acts on different types of receptors and their subtypes. The interaction and integration of biological functions at the level of receptor and postreceptor signaling pathway is an extremely important scientific issue in the regulation of tumor cells by ACh. Because smoking is a high risk factor for lung cancer, the effect of nicotine (nicotine) activating nAChR on lung cancer cells in M1 group was also studied in vitro, and the effects of mAChR on the proliferation and epithelial interstitial transition of lung cancer cells in vitro were also studied. However, the regulation of M 2 mAChR on malignant phenotype of lung cancer in vivo and in vitro has not been reported. Significant progress has been made in targeted treatment of lung cancer, but acquired drug resistance is still a difficult problem. The theory of cancer stem cells holds that drug resistance of lung cancer is closely related to cancer stem cells. The role of choline receptor in molecular targeting drug resistance and dry maintenance of lung cancer stem cells deserves further study. Therefore, this study focuses on the proliferation, metastasis, drug resistance and regulation of lung cancer stem cells by M2 mAChR in vivo and in vitro. Firstly, the presence of non-neurogenic cholinergic system in NSCLC cells A549 / PC9 was detected by CCK-8 RTCA and Western blot. Through the selective antagonists of various subtypes of mAChR on the proliferation of NSCLC cells and the regulation of MAPK and AKT signaling pathway, it was revealed that M2 mAChR played a significant positive regulatory role. M2 mAChR shRNA method further confirmed its role and signal pathway. Tumor-bearing experiment showed that metotrimine mAChR antagonist metotrimine and M2R-shRNA significantly retarded the growth of A549 cells in vivo. Secondly, the inhibition of NSCLC cells A549 and PC9 by MT and M2 mAChR shRNA was investigated by Western blottertranswell and immunofluorescence. MT significantly reversed the occurrence of EMT in A549 cells of NSCLC and reduced the formation of metastatic foci. MT and M2 mAChR siRNA significantly inhibited the expression of NF 魏 B signaling pathway associated with tumor metastasis and its downstream transcription factors snail and zeb1. Thirdly, NSCLC cell line PC9-GR and NSCLC stem cell CD221 -SPC-A1 + M2 mAChR antagonist, which was resistant to EGFR targeting gefitinibor Ge), reversed the expression of M2 mAChR in PC9-GR cell line EMTT CD221 -SPC-A1, and was accompanied by EMT changes (compared with CD221-SPC-A1 cell line). In this study, we first revealed the positive regulation of M2 mAChR on the proliferation, migration and invasion of NSCLC in vivo and in vitro. The results showed that the proliferation was mediated by the PI3K / AKT / MEK / ERK signaling pathway, and the migration and invasion was mediated by NF 魏 B/snail/zeb1 signaling pathway, which may play a role in the production of drug resistance of NSCLC and the dry maintenance of stem cells. These results provide theoretical basis for the regulation mechanism of non-neurogenic cholinergic system on malignant phenotype of lung cancer and the potential application of high selectivity antagonist of M2 subtype.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R734.2

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