【作者】 陈震；

【导师】 马鑫；

【作者基本信息】 江南大学 ， 制药工程与技术， 2017， 博士

【摘要】 近几十年来,由于吸烟率的下降以及阿司匹林的利用率增高、癌症预防知识的广泛传播以及早期检测的推广等因素,结直肠癌发病率和死亡率一直在下降,但它仍是目前最为严重的恶性肿瘤之一。目前在临床上化疗是治疗结直肠癌最有效的一个方法,然而治疗后的远期生存率仍不理想,最主要的原因是患者对化疗药物产生了耐药性。至今为止,对化疗耐药的研究一直都是研究的重点。然而,在长达数十年的针对癌症及癌症耐药的研究中,并没有寻找到明确的发病机制以及精准的耐药分子标志物。因此,探究耐药的分子机制能为结直肠癌的治疗提供一个新方向,并且可以以此分子为靶点,开发新型制剂,设计肠溶性药物靶向给药,以期更好的为临床结直肠癌的治疗提供一种新型药物。本课题发现并研究了影响结直肠癌耐药的一个新的分子机制以及涉及的信号通路,通过大量的临床标本发现,Trp家族中的一个成员TrpC5在结直肠癌标本中异常高表达,我们以此为出发点,探讨TrpC5在结直肠癌发生发展中的作用以及调控机制。主要研究结果如下:RT-PCR结果显示,在31例结直肠癌患者的癌组织和癌旁组织中,TrpC5在癌组织中的表达比对应的癌旁组织显著增高,而TrpC1、TrpC3、TrpC4、TrpC6的转录表达水平没有显著性差异;进一步通过Western blot方法确认TrpC5在癌组织和癌旁组织的表达情况。结果显示,TrpC5在癌组织中的表达水平较对应的癌旁组织有显著性增高,说明TrpC5在结直肠癌中高表达,提示TrpC5在结直肠癌的发生发展中起到了作用。进一步探讨TrpC5与结直肠癌中的临床相关性。通过免疫组化的方法结合临床病理资料,分析TrpC5的表达水平与临床资料的相关性。分析结果发现,TrpC5的表达与肿瘤分级具有显著的正相关(与分化为负相关),而与其他临床因素没有显著性差异;同时,我们发现在结直肠癌新鲜组织原代细胞中,肿瘤分化程度越差,TrpC5的表达及功能水平越强。而后我们在细胞株中探究TrpC5和分化的相关性,通过RT-PCR以及细胞免疫荧光可知,在8株结直肠癌细胞株中,TrpC5的表达水平与分化程度呈负相关,抑制TrpC5表达能降低分化标志物Alp1和Klf4的mRNA水平,以上结果说明在结直肠癌中TrpC5的表达水平与分化相关。结合细胞株模型和临床标本,探究在结直肠癌中TrpC5调节分化程度的分子机制。由RT-PCR结果可知,Wnt所有配体中,仅有Wnt5a的表达与TrpC5的表达以及细胞分化水平相关。进一步,我们发现抑制TrpC5表达后,Wnt5a在细胞浆中表达减少,细胞外液Wnt5a的分泌含量显著减少,β-catenin在细胞核中表达显著降低,发生了细胞核至细胞浆的转移,并且下游靶基因c-myc和Cyclin D1在细胞核中表达也减少,即Wnt/β-catenin通路被抑制;而在高分化细胞株中,过表达TrpC5后,产生了截然相反的结果。在临床标本中,患者肿瘤分化程度越低,癌组织中TrpC5和Wnt5a表达越强,并且血液中Wnt5a的含量也越高。这些结果说明,TrpC5介导Wnt5a的表达进而激活了Wnt/β-catenin信号通路来调节分化。深入分析TrpC5影响肿瘤细胞分化的基础上,是否会影响细胞的干性和药物治疗敏感性。细胞免疫荧光实验以及流式细胞仪检测结果可知,在低分化结直肠癌细胞株中,减少TrpC5表达后,干性标志物CD44以及CD133表达减少;同时我们发现,下调TrpC5的表达,不仅能减弱细胞体外成球能力,也能显著降低细胞体内成瘤能力,即减少TrpC5表达,能减弱细胞的干性。而在耐药方面,Real-time PCR和Western blot实验发现,减少TrpC5表达,能减少耐药蛋白P-gp的转录及翻译表达水平,并且细胞对于化疗药物5-Fu的耐受性减弱;增加TrpC5表达后,耐药蛋白P-gp的表达增强,细胞对5-Fu的耐受性显著增强。以上结果表明,在结直肠癌细胞株中,TrpC5可以通过调节分化进而影响细胞的干性和耐药性。深入研究TrpC5的表达水平在结直肠癌患者中的临床意义,分析其表达水平与患者预后之间的相关性。我们发现TrpC5高表达患者的总生存期和无复发生存期远远低于TrpC5低表达患者,更为重要的是在279例接受了基于5-Fu化疗的患者中,TrpC5高表达组的总生存期和无复发生存期远远比低表达组总生存期短;而在67例未接受基于5-Fu化疗方案的患者中,总生存期和无复发生存期在高表达患者中和低表达患者中没有明显区别。结果提示TrpC5可能是结直肠癌死亡的一个独立的预后因素,并且是临床上对5-Fu化疗方案耐药的一个因素。更多还原

【Abstract】 In recent decades,the incidence and mortality of colorectal cancer(CRC)have been declining due to the decreased smoking rates and the increased use of aspirin and the introduction and dissemination of screening tests.However,CRC is one of the most common forms of malignancy.To date,chemotherapy is the most effective way to treat colorectal cancer.But the long-term survival after treatment is still unsatisfactory.The most important reason is patients resistant to chemotherapy.Although research on resistance has been for several decades,there is no clear mechanism to clarify the resistance to chemotherapy.Thus,exploring molecular mechanism underlying CRC may provide a new direction to CRC treatment.Moreover,it can be used as a target for the development of new types of agents,and design the enteric drug for targeted drug delivery,in order to provide a new drug for better clinical treatment of CRC.In this study,we found a new molecular mechanism and signal pathway that affecting the resistance to CRC.Through a large number of patients with CRC,we found TrpC5,a member of Trp family,was aberrant overexpression in the specimens.Based on these findings,we explored the mechanism of TrpC5 in regulating the development of CRC.The main findings were described as follows:RT-PCR showed that the expression of TrpC5 was significantly higher in cancer samples than in matched adjacent tissue,while TrpC1,TrpC3,TrpC4 and TrpC6 didn’t change.Then we used western bolt to further confirm the expression of TrpC5 in carcinoma and matched adjacent tissue.The results showed that the expression of TrpC5 in carcinoma was higher than in adjacent tissue.These results suggested that TrpC5 plays a role in the development of colorectal cancer.To further explore the clinical relevance of TrpC5 in CRC,immunohistochemistry was used to analysis the relationship between TrpC5 expression and the clinical data.The results showed that the expression of TrpC5 was positively correlated with tumor grading,but had no difference with other clinical factors.While in primary cultures of human CRC cells,we found the expression and the function of TrpC5 were significantly elevated in poorly differentiated CRC cells.Then we investigate the correlation between TrpC5 and differentiation in CRC cell lines.RT-PCR and immunostaining results showed the expression of TrpC5 was negatively with the degree of differentiation in 8 CRC cell lines.Inhibition of TrpC5 reduced the Alp1 and Klf4 expression in m RNA level.These results indicated that the expression of TrpC5 is correlated with differentiation in CRC.Combined with cell lines and clinical samples,we investigate the molecular mechanism of TrpC5 regulating differentiation.RT-PCR showed that in all Wnt ligands,only Wnt5 a expression was correlated with TrpC5 expression and the level of cell differentiation.Furthermore,we found TrpC5 knockdown in poorly differentiated cells decreased Wnt 5a expression and the Wnt5 a secretion and the nuclear translocation of β-catenin,as well as Cyclin D1 and c-myc expression.In contrast,these measures clearly increased when TrpC5 was overexpressed in well differentiated cells.In clinical samples,the lower degree of tumor differentiation,the higher of TrpC5 and Wnt5 a expression in carcinoma,as well as higherconcentrations of Wnt5 a in serum.These results suggested that TrpC5 mediates Wnt5 a expression through Wnt5a/β-catenin signal pathway to regulate differentiation.On the basis of TrpC5 is related to CRC cell differentiation,we further deeply analysis whether TrpC5 will affect cancer cell stemness or drug treatment sensitivity.Immunostaining and FACS showed that inhibited TrpC5 can decrease CD44 and CD133 expression in poorly differentiated cell lines.Moreover,downregulate TrpC5 expression significantly reduced sphere formation and tumor formation in vitro and in vivo.While in terms of resistance,Real-time PCR and western blot showed that inhibited TrpC5 significantly decreased P-gp expression in transcriptional and translational level.Moreover,TrpC5 knockdown in poorly differentiated cells made cells less resistant to 5-Fu.In contrast,overexpression TrpC5 increased P-gp expression and rendered cells less sensitive to 5-Fu.Taken together,these results revealed that TrpC5 regulates differentiation and enhances stemness and drug resistance in CRC cells.To assess the clinical significance of TrpC5 expression in CRC,the survival follow-up information on patients was analyzed.We found patients with tumors that expressed higher levels of TrpC5 showed significantly poorly disease-free and overall survival.More importantly,among the 279 patients who had received 5-Fu-based chemotherapy,patients with tumors that expressed high TrpC5 levels showed less disease-free(DFS)and overall survival(OS)than those with tumors with low TrpC5 expression.In contrast,the remaining67 patients who did not receive 5-Fu-based chemotherapy,showed no difference in DFS and OS regardless of the TrpC5 levels in the tumors.These results provided us initial evidence that TrpC5 is an independent adverse prognostic factor for death in CRC and is a potentially crucial role of TrpC5 in clinical resistance to 5-Fu-based chemotherapy for CRC.更多还原