M3受体亚型在NSCLC细胞生长增殖、侵袭和迁移中的作用及机制研究
发布时间：2018-08-13 20:01
【摘要】：肺癌是发病率和死亡率增长最快,对人群健康和生命威胁最大的恶性肿瘤之一。肺癌最常见的组织学分类为非小细胞肺癌(non-small cell lung cancer,NSCLC)和小细胞肺癌(small cell lung cancer,SCLC),其中80%以上的肺癌病例属于非小细胞肺癌。非小细胞肺癌又可分为鳞状细胞癌(25-30%),腺癌(35-40%)和大细胞癌(10-15%)。早期非小细胞肺癌治疗主要以手术切除为主,常规化疗是联合应用铂类药物(顺铂、卡铂)与抗代谢药、抗微管药或抗拓扑异构酶药,由于毒副反应以及预后不良,仍不能有效提高患者的5年生存率,近年来药物靶向治疗(主要包括以表皮生长因子受体为靶点的酪氨酸激酶抑制剂或单抗,和以肺癌血管生成为靶点的血管内皮生长因子抑制剂或单抗)以及针对患者自身免疫系统的免疫刺激单抗(免疫检查点阻断剂,包括Ipilimumab,Nivolumab,MK-3475等)在特定人群内取得了可喜进展,引发了人们对肺癌发病机制的深入思考和多方位作用靶点的研究,针对其具体的发病机制探索发展新的治疗方法将有望推进肺癌个体化治疗的进程。乙酰胆碱(ACh)是由神经细胞合成的经典神经递质,通过激活乙酰胆碱受体(ACh R)的两个家族成员——毒蕈碱型(m ACh R)和烟碱型(n ACh R)胆碱受体发挥作用。越来越多的研究表明,除神经细胞外,多种肿瘤细胞均可合成和分泌乙酰胆碱作为一种生长因子,参与细胞的生长增殖,抵抗凋亡,血管发生,转移侵袭,促进癌症的进程。这种非神经元胆碱能系统极有可能是调控肿瘤发展进程的另一个重要的信号系统。许多实验室的工作表明,在恶性肿瘤表达的多种m ACh R亚型中,与正常组织相比M3受体表达显著上调,且与肿瘤的预后不良相关。有研究表明,M3受体选择性拮抗剂能够抑制小细胞肺癌细胞增殖并降低裸鼠移植瘤MAPK磷酸化水平,为肺癌的预防和治疗提供了新的药物研发策略。有研究报道,在正常角质化细胞、黑色素瘤细胞、M受体转染的CHO细胞及SCLC细胞上,激活M1或M3受体可改变细胞黏附分子的活性,可能与肿瘤的转移与浸润相关。因此有关M3受体的研究受到极大关注,成为相关研究领域的热点。本研究的重点是观察M3受体拮抗剂对2种NSCLC细胞生长增殖、运动和迁移中的影响,并探索M3受体参与非小细胞肺癌发展进程的信号通路和关键分子,预期研究结果将有助于确认M3受体是否可作为抗肿瘤靶标,将为确立胆碱能受体系统在肿瘤生长调控中的地位提供实验依据。实验方法如下:(1)以CCK-8法测定M3受体拮抗剂、激动剂以及PKC抑制剂对体外培养的NSCLC细胞增殖的影响。(2)应用激光共聚焦显微镜监测激动剂单独给药以及拮抗剂和激动剂伴随给药NSCLC细胞内游离钙离子的动态变化。(3)伤口愈合实验检测M3受体拮抗剂对NSCLC细胞迁移能力的影响。(4)Transwell侵袭实验检测M3受体拮抗剂对NSCLC细胞侵袭能力的影响。(5)利用脂质体介导的si RNA转染技术敲减M3受体并检测敲减M3受体后对NSCLC细胞生长活力的影响。(6)以Western蛋白印迹法检测M3受体拮抗剂对NSCLC细胞增殖信号通路(Akt、MAPK)、细胞周期调节蛋白(cyclin D1、CDK4、p21)以及迁移相关分子(MMP-2)蛋白表达水平的影响。实验结果如下:(1)本研究首先观察M3受体拮抗剂在(1.25μM~80μM)剂量范围内对NSCLC细胞生长增殖的影响,实验结果显示,R2-8018与达非那新体外能够浓度依赖性地抑制H1299细胞和H460细胞的增殖,其中R2-8018抑制H1299细胞增殖的效果更明显,作用48 h的IC50值为10.6μmol/L。(2)外源性补充激动剂卡巴胆碱和氯化乙酰胆碱无法促进H1299细胞和H460细胞增殖。(3)转染M3 si RNA 72 h之后检测检测敲减效率,western蛋白印迹法结果显示,转染M3 si RNA显著降低M3受体的表达(p0.01);CCK-8法48 h和72 h检测结果显示,敲减M3受体,H1299细胞增殖明显被抑制,并且随si RNA作用时间延长,抑制作用更显著(48 h:p0.5,72 h:p0.01)。结果说明,敲减M3受体能够抑制H1299细胞增殖。(4)激光共聚焦显微镜监测细胞内钙离子的动态变化,30μM的卡巴胆碱能够引起H1299细胞钙信号增强,而给予拮抗剂达非那新预处理30 min的H1299细胞30μM卡巴胆碱则观察不到钙信号的增强,达非那新能够完全阻断卡巴胆碱引起的细胞钙信号增强。(5)蛋白激酶C抑制剂Staurosporine体外对H1299细胞存活具有明显的抑制作用,并且R2-8018可浓度依赖性地降低H1299细胞PKC-α蛋白表达(10μM:p0.01,20μM:p0.01)。(6)R2-8018作用H1299细胞24 h,可显著抑制H1299细胞的迁移和侵袭能力,并浓度依赖性地下调基质金属蛋白酶MMP-2蛋白表达水平。(7)达非那新下调H1299细胞Akt、GSK3β和cyclin D1蛋白表达水平。(8)R2-8018上调H1299细胞p21蛋白表达水平。结论:应用M3受体拮抗剂或敲减M3受体能够体外抑制非小细胞肺癌细胞增殖,作为一种G蛋白偶联受体,拮抗M3受体我们可以观察到H1299细胞钙离子释放受到抑制以及蛋白激酶C活性降低,说明拮抗M3受体可能通过其经典的G蛋白偶联信号发挥细胞生长抑制作用;M3受体拮抗剂能降低NSCLC细胞Akt、GSK3β、cyclin D1、CDK4蛋白表达,增加p21蛋白表达,其涉及机制可能为拮抗M3受体可以通过抑制Akt磷酸化,减轻其对下游GSK3β磷酸化,进而增强GSK3β活性,促进对cyclin D1的降解,抑制cyclin D1-CDK4复合物的形成,并且上调p21表达,使细胞无法顺利进入S期而发生G0/G1期阻滞,最终导致细胞增殖受到抑制,此外,M3受体拮抗剂还可能通过抑制MAPK信号通路的激活而产生抑制细胞增殖的作用;M3受体拮抗剂下调基质金属蛋白酶MMP-2蛋白表达,抑制NSCLC细胞的迁移和侵袭能力。
[Abstract]:Lung cancer is one of the malignant tumors with the fastest increase in morbidity and mortality and the greatest threat to human health and life. The most common histological classification of lung cancer is non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). More than 80% of lung cancer cases belong to non-small cell lung cancer. Cellular lung cancer can also be divided into squamous cell carcinoma (25-30%), adenocarcinoma (35-40%) and large cell carcinoma (10-15%). Surgical resection is the main treatment for early non-small cell lung cancer. Conventional chemotherapy is the combination of platinum drugs (cisplatin, carboplatin) and antimetabolic drugs, antimicrotubule drugs or antitopoisomerase drugs, which are still not available due to adverse reactions and poor prognosis. Drug-targeted therapy (mainly tyrosine kinase inhibitors or monoclonal antibodies targeting epidermal growth factor receptors, and vascular endothelial growth factor inhibitors or monoclonal antibodies targeting angiogenesis of lung cancer) and immunostimulatory monoclonal antibodies targeting the patient's autoimmune system (immunoassays) have been effective in improving 5-year survival. Point blockers, including Ipilimumab, Nivolumab, MK-3475 and so on, have made gratifying progress in a specific population, which has triggered in-depth consideration of the pathogenesis of lung cancer and multi-directional target research. Exploring and developing new therapies for the specific pathogenesis of lung cancer will hopefully promote the process of individualized treatment. Acetylcholine (ACh) Acetylcholine receptor (ACh R) and nicotinic choline receptor (NChR), two family members of acetylcholine receptor (ACh R), are classical neurotransmitters synthesized by nerve cells. More and more studies have shown that besides nerve cells, many tumor cells can synthesize and secrete acetylcholine as a growth factor. This non-neuronal cholinergic system is likely to be another important signaling system that regulates the development of tumors. Many laboratory studies have shown that M3 is more likely to be involved in the expression of various subtypes of M ACh R in malignant tumors than in normal tissues. Receptor expression is significantly up-regulated and is associated with poor prognosis. Studies have shown that M3 receptor selective antagonists can inhibit the proliferation of small cell lung cancer cells and reduce MAPK phosphorylation levels in nude mice xenografts, providing a new drug development strategy for the prevention and treatment of lung cancer. Activation of M1 or M3 receptors on cells, M receptor transfected CHO cells and SCLC cells may alter the activity of cell adhesion molecules, which may be related to tumor metastasis and invasion. It is expected that the results will help to confirm whether M3 receptor can be used as an anti-tumor target and provide experimental evidence for the role of cholinergic receptor system in tumor growth regulation. The effects of M3 receptor antagonists, agonists and PKC inhibitors on the proliferation of cultured NSCLC cells were measured by CCK-8 method. (2) The dynamic changes of intracellular free calcium in NSCLC cells treated with agonists alone and with antagonists and agonists were monitored by laser confocal microscopy. (3) Wound healing test was used to detect the effects of M3 receptor antagonists on N. The effect of M3 receptor antagonist on the invasion of NSCLC cells was detected by Transwell invasion assay. (5) The effect of M3 receptor antagonist on the growth of NSCLC cells was detected by liposome-mediated Si RNA transfection and the effect of M3 receptor knockdown on the proliferation of NSCLC cells was detected. (6) Western blotting was used to detect the effect of M3 receptor antagonist on the NSCLC fine cells. The results were as follows: (1) The effects of M3 receptor antagonists on the proliferation of NSCLC cells in the dose range of (1.25 to 80 mu) were observed. The results showed that R2-8018 and R2-8018 were non-specific. Naoxin inhibited the proliferation of H1299 cells and H460 cells in a concentration-dependent manner in vitro, and R2-8018 inhibited the proliferation of H1299 cells more significantly, with the IC50 value of 10.6 micromol/L at 48 h. (2) Exogenous agonists carbachol and acetylcholine chloride could not promote the proliferation of H1299 cells and H460 cells. (3) M3 Si RNA transfection 72 h later. Western blot showed that M3 Si RNA significantly decreased the expression of M3 receptor (p0.01); CCK-8 assay showed that M3 receptor knockdown significantly inhibited the proliferation of H1299 cells, and the inhibitory effect was more significant (48 h: p0.5, 72 h: p0.01). Receptors inhibited the proliferation of H1299 cells. (4) Laser confocal microscopy was used to monitor the dynamic changes of intracellular calcium. Ca2+ signal was enhanced in H1299 cells by carbachol at 30 mu M, but not by carbachol at 30 mu M in H1299 cells pretreated with daphenazine for 30 min. (5) Staurosporine, a protein kinase C inhibitor, significantly inhibited the survival of H1299 cells in vitro, and R2-8018 decreased the expression of PKC-alpha protein in H1299 cells in a concentration-dependent manner (10 mu M: p0.01, 20 mu M: p0.01). (6) R2-8018 significantly inhibited the migration of H1299 cells 24 hours after treatment with R2-8018. Transfer and invasion, and concentration-dependent down-regulation of MMP-2 protein expression. (7) Daphenazine down-regulated the expression of Akt, GSK3 beta and cyclin D1 protein in H1299 cells. (8) R2-8018 up-regulated the expression of p21 protein in H1299 cells. Cell proliferation, as a G-protein-coupled receptor, antagonizes M3 receptor. We can observe the inhibition of calcium release and the decrease of protein kinase C activity in H1299 cells, suggesting that antagonizing M3 receptor may inhibit cell growth through its classical G-protein-coupled signal; M3 receptor antagonist can reduce Akt, GSK3beta, cyclin D1 in NSCLC cells. The expression of CDK4 protein and the expression of p21 protein may be related to the antagonism of M3 receptor by inhibiting Akt phosphorylation, reducing its phosphorylation to downstream GSK3 beta, thereby enhancing the activity of GSK3 beta, promoting the degradation of cyclin D1, inhibiting the formation of cyclin D1-CDK4 complex, and up-regulating the expression of p21, which prevents the cells from entering S phase smoothly. In addition, M3 receptor antagonists may inhibit cell proliferation by inhibiting the activation of MAPK signaling pathway. M3 receptor antagonists down-regulate the expression of MMP-2 protein and inhibit the migration and invasion of NSCLC cells.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2
本文编号：2182029
[Abstract]:Lung cancer is one of the malignant tumors with the fastest increase in morbidity and mortality and the greatest threat to human health and life. The most common histological classification of lung cancer is non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). More than 80% of lung cancer cases belong to non-small cell lung cancer. Cellular lung cancer can also be divided into squamous cell carcinoma (25-30%), adenocarcinoma (35-40%) and large cell carcinoma (10-15%). Surgical resection is the main treatment for early non-small cell lung cancer. Conventional chemotherapy is the combination of platinum drugs (cisplatin, carboplatin) and antimetabolic drugs, antimicrotubule drugs or antitopoisomerase drugs, which are still not available due to adverse reactions and poor prognosis. Drug-targeted therapy (mainly tyrosine kinase inhibitors or monoclonal antibodies targeting epidermal growth factor receptors, and vascular endothelial growth factor inhibitors or monoclonal antibodies targeting angiogenesis of lung cancer) and immunostimulatory monoclonal antibodies targeting the patient's autoimmune system (immunoassays) have been effective in improving 5-year survival. Point blockers, including Ipilimumab, Nivolumab, MK-3475 and so on, have made gratifying progress in a specific population, which has triggered in-depth consideration of the pathogenesis of lung cancer and multi-directional target research. Exploring and developing new therapies for the specific pathogenesis of lung cancer will hopefully promote the process of individualized treatment. Acetylcholine (ACh) Acetylcholine receptor (ACh R) and nicotinic choline receptor (NChR), two family members of acetylcholine receptor (ACh R), are classical neurotransmitters synthesized by nerve cells. More and more studies have shown that besides nerve cells, many tumor cells can synthesize and secrete acetylcholine as a growth factor. This non-neuronal cholinergic system is likely to be another important signaling system that regulates the development of tumors. Many laboratory studies have shown that M3 is more likely to be involved in the expression of various subtypes of M ACh R in malignant tumors than in normal tissues. Receptor expression is significantly up-regulated and is associated with poor prognosis. Studies have shown that M3 receptor selective antagonists can inhibit the proliferation of small cell lung cancer cells and reduce MAPK phosphorylation levels in nude mice xenografts, providing a new drug development strategy for the prevention and treatment of lung cancer. Activation of M1 or M3 receptors on cells, M receptor transfected CHO cells and SCLC cells may alter the activity of cell adhesion molecules, which may be related to tumor metastasis and invasion. It is expected that the results will help to confirm whether M3 receptor can be used as an anti-tumor target and provide experimental evidence for the role of cholinergic receptor system in tumor growth regulation. The effects of M3 receptor antagonists, agonists and PKC inhibitors on the proliferation of cultured NSCLC cells were measured by CCK-8 method. (2) The dynamic changes of intracellular free calcium in NSCLC cells treated with agonists alone and with antagonists and agonists were monitored by laser confocal microscopy. (3) Wound healing test was used to detect the effects of M3 receptor antagonists on N. The effect of M3 receptor antagonist on the invasion of NSCLC cells was detected by Transwell invasion assay. (5) The effect of M3 receptor antagonist on the growth of NSCLC cells was detected by liposome-mediated Si RNA transfection and the effect of M3 receptor knockdown on the proliferation of NSCLC cells was detected. (6) Western blotting was used to detect the effect of M3 receptor antagonist on the NSCLC fine cells. The results were as follows: (1) The effects of M3 receptor antagonists on the proliferation of NSCLC cells in the dose range of (1.25 to 80 mu) were observed. The results showed that R2-8018 and R2-8018 were non-specific. Naoxin inhibited the proliferation of H1299 cells and H460 cells in a concentration-dependent manner in vitro, and R2-8018 inhibited the proliferation of H1299 cells more significantly, with the IC50 value of 10.6 micromol/L at 48 h. (2) Exogenous agonists carbachol and acetylcholine chloride could not promote the proliferation of H1299 cells and H460 cells. (3) M3 Si RNA transfection 72 h later. Western blot showed that M3 Si RNA significantly decreased the expression of M3 receptor (p0.01); CCK-8 assay showed that M3 receptor knockdown significantly inhibited the proliferation of H1299 cells, and the inhibitory effect was more significant (48 h: p0.5, 72 h: p0.01). Receptors inhibited the proliferation of H1299 cells. (4) Laser confocal microscopy was used to monitor the dynamic changes of intracellular calcium. Ca2+ signal was enhanced in H1299 cells by carbachol at 30 mu M, but not by carbachol at 30 mu M in H1299 cells pretreated with daphenazine for 30 min. (5) Staurosporine, a protein kinase C inhibitor, significantly inhibited the survival of H1299 cells in vitro, and R2-8018 decreased the expression of PKC-alpha protein in H1299 cells in a concentration-dependent manner (10 mu M: p0.01, 20 mu M: p0.01). (6) R2-8018 significantly inhibited the migration of H1299 cells 24 hours after treatment with R2-8018. Transfer and invasion, and concentration-dependent down-regulation of MMP-2 protein expression. (7) Daphenazine down-regulated the expression of Akt, GSK3 beta and cyclin D1 protein in H1299 cells. (8) R2-8018 up-regulated the expression of p21 protein in H1299 cells. Cell proliferation, as a G-protein-coupled receptor, antagonizes M3 receptor. We can observe the inhibition of calcium release and the decrease of protein kinase C activity in H1299 cells, suggesting that antagonizing M3 receptor may inhibit cell growth through its classical G-protein-coupled signal; M3 receptor antagonist can reduce Akt, GSK3beta, cyclin D1 in NSCLC cells. The expression of CDK4 protein and the expression of p21 protein may be related to the antagonism of M3 receptor by inhibiting Akt phosphorylation, reducing its phosphorylation to downstream GSK3 beta, thereby enhancing the activity of GSK3 beta, promoting the degradation of cyclin D1, inhibiting the formation of cyclin D1-CDK4 complex, and up-regulating the expression of p21, which prevents the cells from entering S phase smoothly. In addition, M3 receptor antagonists may inhibit cell proliferation by inhibiting the activation of MAPK signaling pathway. M3 receptor antagonists down-regulate the expression of MMP-2 protein and inhibit the migration and invasion of NSCLC cells.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2
【参考文献】
相关期刊论文 前3条
1 邵慧;陈愉生;;非小细胞肺癌侵袭相关的分子学研究进展[J];国际呼吸杂志;2011年10期
2 魏向阳;涂悦;徐忠伟;孙洪涛;令狐海瑞;陈小义;程世翔;张赛;;紫杉醇对U373细胞周期阻滞及增殖抑制的机制研究[J];中国药理学通报;2011年04期
3 王新允,杨菊红,马莹,郑海燕,程波,郑宏伟,朱丛中;肺癌MMPs和TIMPs的表达及浸润转移的研究[J];中国肺癌杂志;2003年04期
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