【作者】 张涛；

【导师】 陈昊；

【作者基本信息】 兰州大学 ， 临床医学·肿瘤学（专业学位）， 2022， 博士

【摘要】 背景胃癌(Gastric cancer,GC)在我国以及甘肃省河西走廊区域属于高发恶性实体瘤。随着手术方式的改良、新型抗肿瘤药物的研发以及综合治疗肿瘤模式的不断完善,GC患者的预后得到了有效的改善。但是,晚期GC患者的五年生存率不容乐观,肿瘤的转移严重阻碍了GC患者的长期生存。因此,从分子层面探索GC细胞发生转移的机制,寻找新的治疗靶点是目前学界关注的焦点。肿瘤细胞的异常增殖以及失巢凋亡抵抗特性,为其发生远处转移奠定了生物学基础。其中,失巢凋亡抵抗作为关键的前步骤,为肿瘤细胞定植于远处靶器官创造了有利条件。对于GC细胞失巢凋亡抵抗分子调控机制的研究,将有助于对GC细胞转移机制的理解。尿激酶型纤溶酶原激活剂受体(Urokinase-type plasminogen activator receptor,PLAUR)在正常组织中几乎不表达,而在肿瘤组织中高表达并与肿瘤细胞的侵袭及迁移有着千丝万缕的关联。在前期的实验中,我们证实了PLAUR在GC中表达上调,并且可能促进了GC细胞失巢凋亡抵抗。其上游的分子调控机制尚不明确。于是,我们通过生物信息学和体内外实验,拟阐明转录因子7样2(Transcription Factor 7 Like 2,TCF7L2)转录激活PLAUR,从而促进GC细胞增殖、失巢凋亡抵抗及迁移。方法(1)通过CCK8、Ed U试剂盒检测TCF7L2、PLAUR对GC细胞增殖的影响;利用超低吸附6孔板构建了失巢凋亡抵抗的细胞模型,通过流式细胞仪和western blot分析TCF7L2、PLAUR对GC细胞失巢凋亡抵抗的影响;通过Transwell和划痕愈合实验评估TCF7L2、PLAUR对GC细胞迁移的影响。(2)利用裸鼠构建皮下成瘤及肺转移模型,以观察TCF7L2和PLAUR对体内肿瘤生长和转移的影响。(3)通过生物信息学的方法查找调控PLAUR表达的潜在转录因子;利用在线数据库分析转录因子TCF7L2在GC中的表达及与预后相关性,并探索TCF7L2在PLAUR启动子区域的结合位点;通过免疫荧光染色对TCF7L2和PLAUR在GC细胞和组织中进行定位和定量表达分析;利用q RT-PCR和western blot分析TCF7L2对PALUR的调控作用;通过双荧光素酶报告基因实验和染色质免疫共沉淀实验确认TCF7L2对PLAUR具有正向的转录调控作用。(4)通过功能回复性实验(Rescue),证实TCF7L2以依赖PLAUR的方式促进GC细胞增殖、失巢凋亡抵抗及迁移。(5)利用免疫组织化学染色和western blot在GC及癌旁组织中检测TCF7L2蛋白的表达情况;基于免疫组化的结果,分析TCF7L2表达与GC患者预后的关系。结果(1)在体外细胞实验中,TCF7L2和PLAUR均提高了GC细胞的增殖、失巢凋亡抵抗以及迁移能力。(2)在体内动物实验中,TCF7L2和PLAUR均促进裸鼠体内肿瘤的生长和转移。(3)经预测转录因子TCF7L2与PLAUR启动子区域存在结合位点,前者主要定位于细胞核和胞浆,后者主要定位于细胞浆中;TCF7L2作为上游分子正向调控了PLAUR在GC中的表达;TCF7L2通过转录激活了PLAUR,促使后者在GC中表达上调。(4)通过Rescue实验,证实了TCF7L2以依赖PLAUR的方式促进GC细胞增殖、失巢凋亡抵抗以及迁移。(5)TCF7L2在GC组织中高表达并与患者预后密切相关,是预测GC患者不良预后的独立危险因子。结论TCF7L2在GC中高表达并且是患者不良预后的独立危险因子;TCF7L2作为转录因子与PALUR启动子相结合,诱导PLAUR在GC中表达上调;同时,TCF7L2以依赖PLAUR的形式促进GC细胞增殖、失巢凋亡抵抗以及迁移。TCF7L2和PLAUR是GC治疗的潜在靶点。更多还原

【Abstract】 BackgroundGastric cancer(GC)is a high-incidence solid tumor in China and the Hexi Corridor Area of Gansu Province.With the improvement of surgical methods,the development of new anti-tumor drugs,and the continuous improvement of comprehensive tumor treatment models,the prognosis of GC patients has been effectively improved.However,the five-year survival rate of patients with advanced GC is still not satisfactory,and the long-term survival of GC patients whose tumor recurrence and metastasis seriously hinder.Therefore,exploring the mechanism of GC cell metastasis from the molecular level and finding effective therapeutic targets is a scientific problem that needs to be solved urgently.The abnormal proliferation of tumor cells and the resistance to anoikis lay the biological foundation for their distant metastasis.Among them,anoikis resistance is a key pre-step,and the colonization of tumor cells in distant target organs creates favorable conditions.The research on the molecular regulation mechanism of GC cell anoikis resistance will help to understand the mechanism of GC cell metastasis.Urokinase-type plasminogen activator receptor(PLAUR)is almost not expressed in normal tissues,but is significantly up-regulated in tumor tissues and is closely related to tumor cell invasion and migration.In previous experiments,we confirmed that PLAUR is up-regulated in GC and may promote anoikis resistance in GC cells.The upstream molecular regulation mechanism is still unclear.Therefore,through bioinformatics and in vivo and in vitro experiments,we clarified that transcription factor 7-like 2(TCF7L2)transcription activates PLAUR,thereby promoting GC cell proliferation,anoikis resistance and migration.Methods(1)The effects of TCF7L2 and PLAUR on the proliferation of GC cells were detected by CCK8 and Ed U kits;a cell model of anoikis resistance was constructed using ultralow adsorption 6-well plate.Then,the effects of TCF7L2 and PLAUR on anoikis resistance of GC cells were analyzed by flow cytometry and western blot;transwell andwound healing experiments were used to evaluate the effect of TCF7L2 and PLAUR on GC cell migration.(2)Use nude mice to inject tumor cells subcutaneously or tail vein to construct subcutaneous tumor formation and lung metastasis models to observe the effects of TCF7L2 and PLAUR on tumor growth and metastasis in vivo.(3)Use bioinformatics methods to find potential transcription factors that regulate PLAUR expression;use online databases to analyze the expression of transcription factor TCF7L2 in GC and its prognosis,and explore the binding site of TCF7L2 in the PLAUR promoter region;Immunofluorescence staining was used to analyze the localization and quantitative expression of TCF7L2 and PLAUR in GC cells and tissues;q RT-PCR and western blot were used to analyze the regulatory effect of TCF7L2 on PALUR;the dual-luciferase reporter gene assay and chromatin immunoprecipitation experiment confirmed that TCF7L2 is positive for PLAUR It has a positive transcriptional regulation effect.(4)Through the functional recovery experiment(Rescue),it is confirmed that TCF7L2 promotes GC cell proliferation,anoikis resistance and migration in a PLAURdependent manner.(5)Immunohistochemistry and western blot were used to analyze the expression of TCF7L2 protein in GC and adjacent tissues;based on the results of immunohistochemistry,the relationship between TCF7L2 and the prognosis of GC patients was analyzed.Results(1)In vitro,TCF7L2 and PLAUR improved the proliferation,anoikis resistance and migration ability of GC cells;(2)In vivo,TCF7L2 and PLAUR promote tumor growth and metastasis in nude mice.(3)It is predicted that there are binding sites between the transcription factor TCF7L2 and the PLAUR promoter region.The former is mainly located in the nucleus and cytoplasm,and the latter is mainly located in the cytoplasm;TCF7L2 as an upstream molecule positively regulates the expression of PLAUR in GC;TCF7L2 activates PLAUR through transcription,prompting the latter to be up-regulated in GC,(4)Through the Rescue experiment,it was confirmed that TCF7L2 promotes GC cell proliferation,anoikis resistance and migration in a PLAUR-dependent manner.(5)TCF7L2 is highly expressed in GC tissues,which is closely related to the prognosis of patients.It is an independent risk factor for predicting the poor prognosis of GCpatients.ConclusionTCF7L2 is highly expressed in GC and is an independent risk factor for poor prognosis of patients.TCF7L2 is used as a transcription factor in combination with the PALUR promoter to induce the up-regulation of PLAUR expression in GC.At the same time,TCF7L2 promotes GC cell proliferation,anoikis resistance and migration in a PLAUR-dependent form.TCF7L2 and PLAUR are potential targets for GC therapy.更多还原