【作者】 王毅；

【导师】 车国卫；

【作者基本信息】 四川大学 ， 外科学（胸心外-普胸）（专业学位）， 2021， 博士

【摘要】 目的:食管癌是具有“中国特色”的消化道恶性肿瘤,不仅食管癌每年新发及死亡人数约占全球一半以上,而且以食管鳞癌为主要病理类型。尽管伴随诊疗技术的进步及新辅助放化疗的推陈出新,但当前食管癌患者的整体5年生存率仍波动于15%至25%之间。靶向治疗的缺乏、放化疗的耐药性以及晚期患者的远处转移是导致食管癌死亡率高的原因。因此,深入解析食管癌发病机制,从分子层面探究其发病机制,寻找新的治疗靶点,才能突破当前食管癌诊疗的瓶颈。癌症的发生发展与其所处的微环境息息相关。肿瘤微环境与肿瘤细胞的彼此促进、共同演化贯穿于肿瘤发生发展的全过程。细胞外基质是肿瘤微环境中最丰富的成分,是由大分子构成的错综复杂的网络,并通过信号转导系统影响细胞的形状、代谢、功能、迁移、增殖和分化等。目前研究认为,细胞外基质蛋白参与构成了肿瘤细胞复杂的交联蛋白网络,在肿瘤的诊断治疗方面具有极高的应用潜力。胶原是细胞外基质的重要组成部分,广泛分布于各个器官和组织中,约占人体蛋白总量的30%。研究发现,胶原伴随着肿瘤的恶性演进而在肿瘤内部和周围蓄积,并由此促进肿瘤生长、转移和药物耐受性的产生。III型胶原蛋白(collagen III)是人类组织中第二大最丰富的胶原蛋白,仅次于I型胶原蛋白,它主要由COL3A1(Collagen alpha-1(III),III型胶原蛋白α1链)基因编码。过去,对COL3A1的研究主要集中在肺、肝血管纤维化领域,仅少数研究报道了III型胶原蛋白的沉积与肿瘤发生发展中的临床相关性研究,尚未就COL3A1在肿瘤恶性演进过程中具体分子作用机制作进一步阐释。故,本课题以COL3A1为研究对象,具体探究其在食管鳞癌的发生、进展中的分子作用机制,以期阐明COL3A1作为食管癌治疗靶点的有效性,为未来食管癌诊断治疗提供重要靶点。材料和方法:1、本研究首先利用实时荧光定量PCR(qPCR,RT-PCR,Quantitative Real-time PCR)法检测5对食管鳞癌及其癌旁上皮组织中COL3A1 mRNA转录水平并用Western Blot检测该5对组织的COL3A1蛋白的翻译水平;然后复苏并传代培养食管鳞癌和正常食管鳞状上皮细胞系,并通过荧光定量PCR(Quantitative Real-time PCR)和Western Blot分别检测COL3A1在食管鳞癌和正常食管中的mRNA转录水平及蛋白翻译水平;收集四川大学华西医院胸外科2009-2011年手术切除的114例食管鳞癌组织样本,并整理相应临床病理信息及预后相关随访资料,将这些患者术中取得的食管鳞癌及其癌旁组织制成相应的组织芯片(TMA),然后再对制备的组织芯片进行免疫组织化学染色实验(Immunohistochemistry,IHC),根据IHC的染色深浅(反应蛋白表达量的高低)和面积进行表达打分,从而通过确定COL3A1蛋白高表达组织和低表达组织的最佳的cut-off值,将患者的性别、年龄、组织学分化程度、肿瘤位置,肿瘤大小与COL3A1蛋白的表达水平等食管鳞癌潜在的预后因素进行单因素和多因素分析,并绘制基线资料表、单因素、多因素分析表和生存曲线,探索COL3A1的表达与食管鳞癌患者临床病理相关指标和患者术后近远期预后的关系;2、COL3A1在食管鳞癌中所调控的功能表型研究,通过体外实验(in vitro)即使用慢病毒分别敲降和过表达相关的食管鳞癌细胞株的COL3A1基因,从而研究COL3A1在食管鳞癌形成中所发挥的功能:(1)细胞增殖:采用CCK-8(cell counting kit-8,细胞计数试剂盒8)法测得的分光光度值(OD)绘制人食管鳞癌细胞株Eca109和KYSE150在COL3A1基因敲降与KYSE180和KYSE510过表达后的72h生长曲线,从而检测COL3A1基因对食管鳞癌细胞体外增殖能力的调节作用。并进一步通过平板克隆实验和肿瘤球实验检测COL3A1基因对食管鳞癌细胞较长期和体内模拟三维环境中增殖能力的调控作用。(2)细胞迁移:通过细胞划痕实验(Wound healing assay)和Transwell实验评估COL3A1基因敲降和过表达后食管鳞癌细胞系迁移或侵袭能力的改变,以探索COL3A1基因对食管鳞癌远处转移能力的调节作用;(3)细胞凋亡:通过Annexin V/PI双染色法研究COL3A1基因是否对食管鳞癌细胞株的早期和晚期凋亡存在一定的调节作用;(4)动物实验:构建食管鳞癌细胞COL3A1基因敲降的稳转株,进行免疫缺陷鼠皮下成瘤实验,以验证体内COL3A1基因是否存在体外实验中对食管鳞癌细胞增殖的影响;3、利用mRNA测序技术(mRNA sequencing,转录组测序)检测食管鳞癌Eca109细胞COL3A1基因敲降细胞和正常对照细胞内mRNA转录水平的变化情况,进而通过GO、KEGG功能富集分析和GSEA基因集富集分析总结COL3A1下游可能的信号通路及蛋白。最后通过WB检测细胞相关差异基因表达的情况。结果:1、COL3A1在食管鳞癌中的表达水平及其临床意义实时荧光定量PCR(Quantitative Real-time PCR)和蛋白印迹(WB)结果证明食管鳞癌组织中COL3A1 mRNA在转录水平上和显著高于配对的癌旁组织,且COL3A1蛋白在翻译水平上也高于配对的癌旁组织。并且在食管鳞癌细胞系中,COL3A1 mRNA转录水平和蛋白翻译水平也均高于食管正常鳞状上皮细胞系。食管鳞癌中COL3A1蛋白的表达水平与患者的年龄、肿瘤位置、大小和肿瘤的分化程度无明显相关性,而与患者的性别、肿瘤的p T分期呈明显正相关。根据患者COL3A1蛋白在食管鳞癌组织中表达情况将患者分为COL3A1高表达和低表达两组,通过Log-rank检验及COX比例风险模型,COL3A1低表达组食管鳞癌患者的生存期明显比高表达组患者更长(P<0.05),且多因素分析提示COL3A1表达水平是食管鳞癌患者预后的独立危险因素(P<0.05)。2、COL3A1基因表达水平在食管鳞癌增殖和侵袭呈正相关关系通过CCK-8绘制的72h细胞生长曲线及细胞平板克隆实验证实,敲降COL3A1基因的食管鳞癌细胞系增殖能力显著降低,COL3A1基因过表达能进一步增高食管鳞癌细胞系的增殖能力;通过细胞划痕实验和Transwell实验发现,敲降COL3A1基因的食管鳞癌细胞迁移能力明显降低,而COL3A1基因过表达增强了食管鳞癌细胞系的迁移;通过Annexin V/PI双染色法并通过流式细胞仪对荧光进行分析发现COL3A1基因敲降一定程度上促进了食管鳞癌细胞的凋亡,但差异无统计学意义。与体外实验相呼应,裸鼠皮下成瘤模型显示COL3A1的敲降显著降低了食管鳞状细胞移植瘤的成瘤能力。3、COL3A1基因调控食管鳞癌增殖及迁移的下游通路机制借助mRNA sequencing技术,在食管鳞癌Eca109细胞株中对比COL3A1敲降组(sh COL3A1)与正常对照细胞(NC)的mRNA转录水平的差异情况,进而通过GO、KEGG和GSEA基因富集分析预测COL3A1下游可能的信号通路。最后通过WB验证了NF-kappa B信号通路与COL3A1的正向调控关系。结论:本研究证实了COL3A1在食管鳞癌中呈高表达,且COL3A1的表达量与食管鳞癌患者的p T分期呈正相关,与患者的预后呈负相关。COL3A1的生物学功能主要是促进食管鳞癌细胞增殖和迁移。进一步的机制研究表明:COL3A1基因通过激活下游NF-kappa B通路相关蛋白p-Ik B和P65调控食管鳞癌细胞的增殖和迁移。综上,COL3A1可能是食管鳞癌的一个肿瘤学分子标志物和潜在的治疗靶点。更多还原

【Abstract】 Objective:Esophageal cancer is a malignant tumor of the digestive tract with "Chinese characteristics".The number of new cases and deaths of esophageal cancer accounts for more than half of the global total every year,and esophageal squamous cell carcinoma is the main pathological type.Despite advances in diagnosis and treatment techniques and the introduction of neoadjuvant chemoradiotherapy,the overall5-year survival rate of esophageal cancer patients fluctuates between 15% and 25%.The lack of targeted therapy,chemoradiotherapy resistance and distant metastasis in advanced patients are the reasons for the high mortality rate of esophageal cancer.Therefore,only by deeply analyzing the pathogenesis of esophageal cancer,exploring its pathogenesis from the molecular level,and looking for new therapeutic targets,can we break through the bottleneck of the current diagnosis and treatment of esophageal cancer.The occurrence and development of cancer is closely related to its microenvironment.The mutual promotion and co-evolution of tumor microenvironment and tumor cells runs through the whole process of tumor genesis and development.Extracellular matrix(ECM)is the most abundant component in the tumor microenvironment.It is a complex network composed of macromolecules that influence cell shape,metabolism,function,migration,proliferation and differentiation through signal transduction system.Current studies suggest that extracellular matrix proteins are involved in the complex cross-linking protein network of tumor cells,and have a high application potential in the diagnosis and treatment of tumors.Collagen,an important component of extracellular matrix,is widely distributed in various organs and tissues,accounting for about 30% of the total protein in the human body.It has been found that collagen accumulates in and around tumors along with the malignant progression of tumors,and thus promotes tumor growth,metastasis and drug tolerance.Collagen III is the second most abundant collagen in human tissues,second only to type I collagen,which is mainly encoded by COL3A1(Collagen alpha-1)gene.In the past,studies on COL3A1 mainly focused on pulmonary and hepatic vascular fibrosis,and only a few studies reported the clinical correlation between the deposition of type III collagen and the development of tumors,and the specific molecular mechanism of COL3A1 in the malignant progression of tumors has not been further explained.Therefore,this study took COL3A1 as the research object to specifically explore its molecular mechanism of action in the occurrence and progression of esophageal squamous cell carcinoma,with a view to clarifying the effectiveness of COL3A1 as a therapeutic target for esophageal cancer and providing an important target for the diagnosis and treatment of esophageal cancer in the future.Materials and methods:1.In this study,Quantitative real-time PCR(q PCR,RT-PCR,Quantitative real-time PCR)was used to detect the transcription level of COL3A1 mRNA in 5pairs of esophageal squamous cell carcinoma and its adjacent epithelial tissues,and Western Blot was used to detect the translation level of COL3A1 protein in these 5pairs of tissues.Then,the cell lines of esophageal squamous cell carcinoma and normal esophageal squamous cell carcinoma were resuscitated and subcultured.Quantitative real-time PCR and Western Blot were used to detect the mRNA transcription levels and protein translation levels of COL3A1 in esophageal cancer and normal esophagus,respectively.Collection of thoracic surgery,west China hospital of sichuan university 2009-2011,114 cases of surgical removal of the esophageal squamous carcinoma tissue samples,and the corresponding clinical pathological information follow-up and prognosis related information,these patients who get the esophageal squamous carcinoma and made the corresponding tissue adjacent to carcinoma tissue microarray(TMA),and then carried out on the preparation of tissue microarray immunohistochemical staining,Immunohistochemistry,IHC)experiment,according to the dyeing depth of IHC(c-reactive protein table a large amount of high and low)to express and area,Thus by determining COL3A1 high protein expression and lower expression tissue of the best values of the cut-off,will the patient’s gender,age,degree of histological differentiation,tumor location,tumor size and COL3A1 protein expression level of esophageal squamous cell carcinomas potential prognostic factors,single factor and multiple factors analysis,and draw the baseline data sheet,the single factor and multiple factors analysis table and survival curve,explore the expression of COL3A1 related to esophageal squamous carcinoma in patients with clinical pathological indexes and the relationship between the prognosis of patients with postoperative Jin Yuan Qi;2.Study on the functional phenotypes regulated by COL3A1 in esophageal squamous cell carcinoma.Through in vitro experiment(in vitro),i.e.using lentivirus to knock down and overexpress the COL3A1 gene of related esophageal squamous cell carcinoma lines,the function of COL3A1 in the formation of esophageal squamous cell carcinoma was studied:(1)Cell proliferation: The 72 h growth curve of human esophageal squamous cell cell lines ECA109 and KYSE150 after COL3A1 gene knockdown and KYSE180 and KYSE510 overexpression was plotted by using the spectrometric value(OD)measured by CCK-8(Cell counting kit 8)method,so as to detect the regulatory effect of COL3A1 gene on the proliferation ability of esophageal squamous cell cell cells in vitro.Furthermore,plate cloning assay and tumor ball assay were used to detect the regulation effect of COL3A1 gene on the proliferation of esophageal squamous cell carcinoma cells in a long-term and three-dimensional environment in vivo.(2)Cell migration: The change of migration or invasion ability of esophageal squamous cell carcinoma lines after COL3A1 gene knockdown and overexpression was evaluated by Wound healing assay and Transwell assay to explore the regulatory effect of COL3A1 gene on distant metastasis of esophageal squamous cell carcinoma.(3)Cell apoptosis: Annexin V/PI double staining was used to study whether COL3A1 gene could regulate the early and late apoptosis of esophageal squamous cell carcinoma cell lines.(4)Animal experiments: stable transgenic strains of knockdown COL3A1 gene in esophageal squamous cell carcinoma cells were constructed to conduct subcutaneous tumor-forming experiments in immunodeficient mice to verify whether COL3A1 gene in vivo has an effect on the proliferation of esophageal squamous cell carcinoma cells in vitro.3.The changes of mRNA transcription level in COL3A1 knockdown cells of esophageal squamous cell carcinoma ECA109 cells and normal control cells were detected by mRNA sequencing(sequencing,transcriptome sequencing),and the possible downstream signaling pathway and protein of COL3A1 were summarized by GO and KEGG function enrichment analysis and GSEA gene set enrichment analysis.Finally,cell differential gene expression was detected by WB.Results:1.Expression level and clinical significance of COL3A1 in esophageal squamous cell carcinomaQuantitative real-time PCR and western blot(WB)results showed that the transcription level of COL3A1 mRNA in esophageal squamous cell carcinoma tissues was significantly higher than that in the paired paracancerous tissues,and the translation level of COL3A1 protein was also higher than that in the paired paracancerous tissues.Moreover,the mRNA transcription level and protein translation level of COL3A1 in esophageal squamous cell cell lines were higher than those in normal esophageal squamous cell lines.The expression level of COL3A1 protein in esophageal squamous cell carcinoma was not significantly correlated with the patient’s age,tumor location,tumor size and tumor differentiation degree,but was significantly positively correlated with the patient’s gender and the PT stage of the tumor.According to patients’ COL3A1 protein expression in esophageal squamous carcinoma tissues divided the patients into COL3A1 high expression and low expression in the two groups,through the Log-rank test and COX proportional hazards model,COL3A1 low expression group of patients with esophageal squamous cell carcinomas longer survival expression is significantly higher than the group of patients(P < 0.05),and the multi-factor analysis COL3A1 expression levels are independent risk factors for the prognosis of patients with esophageal squamous cell carcinomas(P < 0.05).2.The expression level of COL3A1 gene was positively correlated with the proliferation and invasion of esophageal squamous cell carcinoma72h cell growth curve drawn by CCK-8 and cell plate cloning experiments confirmed that the proliferation ability of esophageal squamous cell cell lines knocked down COL3A1 gene was significantly reduced,and the overexpression of COL3A1 gene could further increase the proliferation ability of esophageal squamous cell cell lines.Cell scratch assay and Transwell assay showed that the migration ability of esophageal squamous cell carcinoma cells was significantly reduced when COL3A1 gene was knocked down,while the overexpression of COL3A1 gene enhanced the migration of esophageal squamous cell carcinoma lines.Fluorescence analysis by Annexin V/PI double staining and flow cytometry showed that COL3A1 knockdown promoted apoptosis of esophageal squamous cell carcinoma cells to a certain extent,but the difference was not statistically significant.Consistent with in vitro experiments,subcutaneous tumorigenesis model of nude mice showed that COL3A1 knockdown significantly reduced the tumorigenesis ability of esophageal squamous cell transplantation tumor.3.The downstream pathway mechanism of COL3A1 gene regulating proliferation and migration of esophageal squamous cell carcinomaWith the help of mRNA sequencing technology,the difference of mRNA transcription level between the COL3A1 knockdown group(SHCOL3A1)and the normal control cells(NC)was compared in ECA109 cell line of esophageal squamous cell carcinoma,and the possible downstream signaling pathway of COL3A1 was predicted through the enrichment analysis of GO,KEGG and GSEA genes.Finally,the positive regulatory relationship between the NF-kappa B signaling pathway and COL3A1 was verified by WB.Conclusion:This study confirmed that COL3A1 is highly expressed in esophageal squamous cell carcinoma,and the expression level of COL3A1 is positively correlated with the p T staging of patients with esophageal squamous cell carcinoma,and negatively correlated with the prognosis of patients.The biological function of COL3A1 is mainly to promote the proliferation and migration of esophageal squamous cell carcinoma cells.Further mechanism studies showed that COL3A1 regulates proliferation and migration of esophageal squamous cell carcinoma cells through activation of downstream NF-kappa B pathway related proteins p-Ikb and P65.In conclusion,COL3A1 may be an oncologic molecular marker and a potential therapeutic target for esophageal squamous cell carcinoma.更多还原