负性共刺激分子B7-H3通过c-Met途径对结直肠癌EMT的作用及临床意义

发布时间：2018-08-27 17:22

【摘要】：结直肠癌是严重威胁人类健康的重大疾病。转移是导致结直肠癌预后不良的关键问题,超过三分之一的结直肠癌患者最终会发生转移,给临床治疗带来极大困难,严重影响患者的疗效和生存质量。因此,结直肠癌治疗的难题在于转移,必须寻找可以有效干预结直肠癌转移的分子靶点,而这有赖于结直肠癌转移发生机制研究的突破。然而,结直肠癌的转移是涉及多个步骤的连续过程,极其复杂,其具体机制目前仍不清楚。肿瘤细胞之间存在明显的异质性,并非所有肿瘤细胞都能发生转移,只有那些获得一定迁移和侵袭能力的肿瘤细胞才能突破重重障碍,形成远隔组织或器官转移灶。研究表明,上皮-间质转化(Epithelial-mesenchymal transition,EMT)是导致肿瘤细胞发生侵袭和转移的重要原因,是肿瘤发生转移的关键启动步骤。在EMT启动肿瘤细胞侵袭转移的过程中,播撒的肿瘤细胞获得了类似肿瘤干细胞自我更新的特性,进而形成了肉眼可见的转移灶。近年随着对肿瘤免疫研究的不断深入,一组介导免疫调节的重要分子——负性B7家族分子:PD-L1(B7-H1)/PD-1、B7-H3、B7-H4和CTLA-4以及Tim-3等,异常表达在一系列肿瘤组织和/或免疫细胞,参与肿瘤免疫逃逸、肿瘤侵袭转移等过程,并与肿瘤临床病理和预后密切相关。其中,抗CTLA-4、PD-1及PD-L1单抗在多种肿瘤治疗中显示出卓著疗效,负性B7家族分子在肿瘤免疫治疗中的前景受到空前关注。B7-H3是2001年从人DC的cDNA库中克隆得到的一个B7家族成员。人B7-H3基因定位于15q24,属免疫球蛋白家族的I型跨膜蛋白。迄今为止,已先后发现B7-H3蛋白在诸多肿瘤组织中高表达,包括胃癌、肺癌、前列腺癌等。B7-H3在人类肿瘤组织的异常表达,并参与了肿瘤的发生和发展,但是B7-H3参与肿瘤生长与转移的作用机制尚不完全明确。本研究所一直致力于负性B7家族分子(PD-L1、B7-H3、B7-H4、CTLA-4和Tim-3)与肿瘤免疫的研究,近来在结合蛋白研究方面取得部分进展,通过质谱分析找到了可以与b7-h3相互作用的原癌基因编码蛋白c-met,鉴此为本课题展开奠定了良好的基础。c-met在多种肿瘤中高表达并异常激活,在肿瘤发生发展、侵袭转移等多个环节均发挥关键作用。鉴此,本文以人结直肠癌为研究对象,b7-h3和c-met相互作用为主线,通过临床标本和体内外实验多个层面的研究,进一步探讨b7-h3调控结直肠癌emt促进转移的作用机制及临床意义。第一部分负性共刺激分子b7-h3在结直肠癌中的表达及临床意义【目的】分析人结直肠癌细胞株、人结直肠癌新鲜组织以及石蜡组织中共刺激分子b7-h3的表达,进而探讨b7-h3与结直肠癌临床病理因素、患者预后的关系,揭示b7-h3在结直肠癌组织标本的异常表达及其临床意义。【方法】分别从mrna和蛋白水平分析了6株结直肠癌细胞株中b7-h3的表达,并同时观察了肿瘤细胞上清中可溶性b7-h3的表达;进一步比较分析了10例手术切除的结直肠癌肿瘤组织和远端正常肠组织标本中b7-h3的mrna和蛋白水平的表达差异;进而通过免疫组化方法分析了197例配对的结直肠癌癌组织和癌旁组织中b7-h3的表达,并结合病理资料及生存时间,统计分析b7-h3表达的临床意义。【结果】(1)6株结直肠癌细胞表面均存在不同程度膜型b7-h3的表达,同时还可分泌可溶性b7-h3;(2)较之于远端正常肠组织,手术切除的新鲜结直肠癌肿瘤组织中b7-h3呈高表达,免疫组化分析石蜡组织结果显示:197例结直肠癌患者中,73例患者组织低表达b7-h3分子,占37.1%;124例患者组织高表达b7-h3分子,占62.9%;(3)结合患者的术后总体生存时间和无病生存时间进行分析,发现b7-h3表达的高低与患者的预后密切相关(p0.01),较之于b7-h3低表达的患者,b7-h3高表达患者的总体生存时间与无病生存时间都大幅下降;(4)进一步的统计发现,b7-h3表达高低与患者的duke's分期相关(p0.05),而与性别、年龄、肿瘤大小、肿瘤分期和淋巴结转移等无统计学差异;(5)随着b7-h3表达水平的升高,患者发生远处转移与b7-h3表达水平的呈相关性上升。【结论】共刺激分子b7-h3在结直肠癌细胞、新鲜组织以及石蜡组织中异常高表达,并与患者的duke's分期和不良预后密切相关,提示了b7-h3在结直肠癌的进展与转移中发挥重要作用。第二部分b7-h3在结直肠癌细胞的emt及干性维持中的作用【目的】通过分析b7-h3对肿瘤细胞迁移、侵袭能力以及干性的作用,以期明确b7-h3在结直肠癌细胞emt和干性维持中的调控作用。【方法】以高表达b7-h3的结直肠癌细胞系hct116、sw480作为研究对象,经sirna干扰下调b7-h3的表达后,通过real-timepcr、westernblot和免疫荧光法观察肿瘤细胞中emt相关标志分子、转录因子和干性基因的mrna和蛋白水平表达的变化;进一步以沉默或过表达b7-h3的hct116、sw480为研究对象,通过迁移实验、划痕实验和侵袭实验观察肿瘤细胞体外迁移和侵袭的能力;通过体外细胞球实验和化疗药抵抗实验,以及皮下接种肿瘤模型分析b7-h3在肿瘤细胞干性维持中的作用。【结果】(1)b7-h3表达下调后,结直肠癌细胞hct116、sw480中的上皮标志e-cadherin呈上调表达,间质标志vimentin和n-cadherin呈下调表达,且emt相关转录因子snail、slug、zeb1、twist的表达也显著下调;(2)b7-h3表达下调后,肿瘤细胞中干性基因(cd133、nanog、bmi1、lgr5、abcb1、abcg2、oct4)呈下调表达;(3)进一步的迁移实验、划痕实验和侵袭实验结果显示,b7-h3表达下调后,结直肠癌细胞hct116、sw480的体外迁移和侵袭能力受到抑制;但过表达b7-h3后,结直肠癌细胞hct116、sw480的体外迁移和侵袭能力又得到增强;(4)体外细胞球实验发现:b7-h3表达下调后,结直肠癌细胞hct116、sw480的细胞球形成能力受到抑制;(5)化疗药抵抗实验进一步揭示了b7-h3表达下调后,结直肠癌细胞hct116、sw480的对化疗药更为敏感;(6)通过荷瘤小鼠体内实验证实,结直肠癌细胞hct116中b7-h3表达下调后,其在小鼠体内形成肿瘤的体积及数量均显著降低。【结论】共刺激分子b7-h3表达沉默后,结直肠癌细胞的间质化转变受阻,进而肿瘤细胞的体外迁移和侵袭能力受到抑制。同时,结直肠癌细胞的干性标志表达也显著下调,进而其体外成球和化疗药抵抗能力减弱。此外,结直肠癌细胞的体内成瘤能力也显著降低。第三部分b7-h3、e-cadherin及c-met在结直肠癌中的共表达及临床意义【目的】在发现了b7-h3可与原癌基因编码蛋白c-met相互作用,以及明确了b7-h3对emt调控作用的基础上,通过分析结直肠癌组织中b7-h3、上皮标志e-cadherin及c-met的表达,探讨三者与结直肠癌临床病理因素、患者预后的关系,揭示b7-h3、e-cadherin及c-met共表达在结直肠癌发生发展及预后中的作用。【方法】收集197例手术切除的结直肠癌癌组织和癌旁组织,通过免疫组化方法分析了197例配对组织中b7-h3、e-cadherin及c-met的表达,进一步结合病理资料及生存时间,统计分析b7-h3、e-cadherin及c-met表达的相关性及临床意义。【结果】(1)免疫组化结果显示,197例结直肠癌患者中,31.5%组织标本中e-cadherin正常表达于细胞膜,而68.5%组织标本中e-cadherin异常表达于细胞浆或不表达;(2)71.1%组织标本中异常表达c-met,28.9%组织标本中不表达c-met;(3)进一步统计发现,结直肠癌肿瘤组织中异常表达的b7-h3和膜e-cadherin之间呈负相关的关系,且高表达b7-h3和浆e-cadherin的患者的总体生存时间与无病生存时间都大幅下降;(4)异常表达的b7-h3和c-met之间呈正相关的关系,且具有显著统计学差异。【结论】在结直肠癌肿瘤组织中异常高表达b7-h3、浆e-cadherin和c-met,b7-h3与膜e-cadherin之间呈负相关,与c-met之间呈正相关,显著影响患者的总体生存时间和无病生存时间。这些均提示b7-h3与c-met相互作用后可参与结直肠癌emt的调控。第四部分b7-h3通过c-met途径对结直肠癌细胞emt的调控机制【目的】通过探讨b7-h3与c-met相互作用后在结直肠癌细胞emt中的作用机制,为b7-h3促进人结直肠癌的进展和转移提供理论依据。【方法】以过表达b7-h3的结直肠癌细胞系sw480作为研究对象,通过免疫共沉淀和westernblot方法观察结直肠癌细胞中b7-h3和c-met的相互作用;通过共聚焦显微镜观察结直肠癌细胞中b7-h3和c-met的共定位。以低表达b7-h3的结直肠癌细胞rko为研究对象,进行b7-h3过表达和c-met沉默共处理后,通过迁移实验和划痕实验分析b7-h3/c-met对肿瘤细胞迁移侵袭能力的调控作用,通过干性基因检测和细胞球实验探讨b7-h3/c-met对肿瘤细胞干性维持的影响。在此基础上,外源加入人重组b7-h3蛋白观察c-met、p-met的表达变化;进一步通过westernblot观察b7-h3表达沉默后结直肠癌细胞中c-met下游关键信号分子akt、erk磷酸化水平以及emt转录因子snail的表达变化。【结果】(1)在过表达b7-h3的sw480细胞中共刺激分子b7-h3可与c-met相关作用,通过共聚焦显微镜可观察到两者存在共定位;(2)较之于对照组,b7-h3过表达组的肿瘤细胞表面干性标志表达明显上调,且迁移、划痕愈合和细胞球形成能力显著增强;而较之于b7-h3过表达组,b7-h3过表达/c-met沉默表达共处理组的肿瘤细胞表面干性标志表达明显下调,同时迁移、划痕愈合和细胞球形成能力也显著减弱;(3)外源加入的人重组蛋白b7-h3可上调p-met的表达水平;(4)当b7-h3表达沉默后会影响下游关键信号分子akt、erk的磷酸化水平以及emt关键转录因子snail的表达。【结论】共刺激分子b7-h3可与c-met相互作用,其后通过影响p-met的表达,进而通过下游信号轴akt/snail参与调控结直肠癌细胞的emt,从而影响肿瘤的转移。综上所述,本课题通过临床标本分析和体内外多个层面和不同水平的实验证实共刺激分子b7-h3与结直肠癌临床病理、进展和转移密切相关,继而发现b7-h3通过emt机制加速结直肠癌细胞发生间质化转变,进而促进肿瘤的进展与转移,b7-h3分子有望成为监测结直肠癌转移的有价值的生物标志。更为重要的是本文首次发现结直肠癌细胞表达b7-h3可以与原癌基因编码蛋白c-met相互作用,促进c-Met信号及相应生物学功能,进而阐明了B7-H3调控结直肠癌EMT进程促进肿瘤进展与转移的作用机制,为结直肠癌转移的免疫干预提供一个新的靶点。
[Abstract]:Colorectal cancer is a serious threat to human health. Metastasis is a key problem leading to poor prognosis of colorectal cancer. More than one third of colorectal cancer patients will eventually metastasize, which brings great difficulties to clinical treatment and seriously affects the efficacy and quality of life of patients. However, the metastasis of colorectal cancer is a continuous process involving multiple steps, and its specific mechanism is still unclear. There is obvious heterogeneity between tumor cells, not all tumor details. Only those tumor cells with certain ability of migration and invasion can break through barriers and form distant tissue or organ metastasis foci. Studies have shown that epithelial-mesenchymal transition (EMT) is an important cause of invasion and metastasis of tumor cells, and metastasis of tumor. In the process of EMT initiating invasion and metastasis of tumor cells, seeded tumor cells acquire self-renewal characteristics similar to that of tumor stem cells, thus forming metastatic foci visible to the naked eye. L1 (B7-H1) / PD-1, B7-H3, B7-H4, CTLA-4 and Tim-3 are abnormally expressed in a series of tumor tissues and / or immune cells, which are involved in tumor immune escape, tumor invasion and metastasis, and are closely related to the clinical pathology and prognosis of the tumor. Among them, anti-CTLA-4, PD-1 and PD-L1 monoclonal antibodies have shown remarkable therapeutic effects in a variety of tumor treatments, and negative B7 have been found. B7-H3 is a member of the B7 family cloned from the human DC cDNA Library in 2001. The human B7-H3 gene is located at 15q24 and belongs to the immunoglobulin family I transmembrane protein. B7-H3 is abnormally expressed in human tumor tissues and participates in the occurrence and development of tumor. However, the mechanism of B7-H3 involved in tumor growth and metastasis is still unclear. Some progress has been made in this field. The proto-oncogene coding protein c-met, which can interact with b7-h3, has been found by mass spectrometry analysis. It has laid a good foundation for this research. c-met is highly expressed and abnormally activated in various tumors, and plays a key role in the development, invasion and metastasis of tumors. The interaction between B7-H3 and c-Met in human colorectal cancer was the main clue. the mechanism and clinical significance of B7-H3 regulating the metastasis of colorectal cancer by EMT were further explored through clinical specimens and in vivo and in vitro experiments. part one: the expression and clinical significance of negative costimulatory molecule B7-H3 in colorectal cancer [Methods] To analyze the expression of costimulatory molecule B7-H3 in human colorectal cancer cell lines, fresh colorectal cancer tissues and paraffin tissues, and to explore the relationship between B7-H3 and clinicopathological factors, prognosis of colorectal cancer patients, and to reveal the abnormal expression of B7-H3 in colorectal cancer tissues and its clinical significance. The expression of B7-H3 was analyzed in 6 colorectal cancer cell lines, and the expression of soluble B7-H3 in the supernatant of tumor cells was also observed. The expression of B7-H3 mRNA and protein in 10 colorectal cancer tissues and distal normal intestinal tissues were compared and analyzed by immunohistochemistry. The expression of B7-H3 in colorectal cancer tissues and adjacent tissues of 97 matched patients was analyzed by pathological data and survival time. The expression of B7-H3 was high in fresh colorectal cancer tissues after operation. The results of immunohistochemical analysis showed that in 197 colorectal cancer patients, 73 cases had low expression of b7-h3, accounting for 37.1%; 124 cases had high expression of b7-h3, accounting for 62.9%; (3) combined with the overall survival time and disease-free survival time of the patients after operation. It was found that the expression of B7-H3 was closely related to the prognosis of patients (p0.01). Compared with the patients with low expression of b7-h3, the overall survival time and disease-free survival time of patients with high expression of B7-H3 decreased significantly. (4) Further statistics showed that the expression of B7-H3 was related to the duke's stage of patients (p0.05), but with gender, age, tumor size, and so on. There was no significant difference in tumor stage and lymph node metastasis. (5) With the increase of B7-H3 expression level, distant metastasis and B7-H3 expression level were significantly correlated. Part two: the role of B7-H3 in EMT and dry maintenance of colorectal cancer cells [Methods] The expression of EMT-related markers, transcription factors and dry gene mRNA and protein in colorectal cancer cell lines HCT116 and SW480 were detected by real-time pcr, Western blot and immunofluorescence after siRNA interference. One step was to observe the ability of tumor cells to migrate and invade in vitro by migration test, scratch test and invasion test, and to analyze the role of B7-H3 in the maintenance of tumor cell stem by cell ball test in vitro, chemotherapeutic drug resistance test and subcutaneous inoculation tumor model. Results: (1) after B7-H3 expression was down-regulated, the expression of e-cadherin, the epithelial marker of HCT116 and sw480, vimentin and n-cadherin, and the expression of snail, slug, ZEB1 and twist were down-regulated significantly; (2) after B7-H3 expression was down-regulated, the trunk genes (cd133, nanog, bmi1, lgr5, ab) were down-regulated. Cb1, abcg2, Oct4 were down-regulated; (3) further migration test, scratch test and invasion test showed that the down-regulated expression of B7-H3 inhibited the migration and invasion ability of colorectal cancer cells hct116, SW480 in vitro; but after overexpression of b7-h3, the migration and invasion ability of colorectal cancer cells hct116, SW480 in vitro was enhanced; (4) in vitro migration and invasion ability of colorectal cancer cells hct116, SW480 was enhanced; (4) in vitro migration and invasion ability of colorectal cancer cells SW48 The results of cell sphere assay showed that the sphere formation ability of colorectal cancer cells HCT116 and SW480 was inhibited after the down-regulation of B7-H3 expression; (5) chemotherapeutic drug resistance assay further revealed that after the down-regulation of B7-H3 expression, colorectal cancer cells HCT116 and SW480 were more sensitive to chemotherapeutic drugs; (6) in vivo experiments in tumor-bearing mice confirmed that B in colorectal cancer cells HCT116 was more sensitive to chemotherapeutic drugs. [Conclusion] When the expression of costimulatory molecule B7-H3 was silenced, the stromal transformation of colorectal cancer cells was blocked, and the migration and invasion of colorectal cancer cells were inhibited in vitro. The co-expression of b7-h3, E-cadherin and c-Met in colorectal cancer and its clinical significance [Objective] It was found that B7-H3 could interact with c-met, a proto-oncogene coding protein, and the relationship between B7-H3 and EMT was clarified. On the basis of regulation, the expression of b7-h3, E-cadherin and c-Met in colorectal cancer tissues was analyzed to explore the relationship between the expression of b7-h3, E-cadherin and c-Met and the clinicopathological factors and prognosis of colorectal cancer. The role of co-expression of b7-h3, E-cadherin and c-Met in the occurrence, development and prognosis of colorectal cancer was revealed. The expressions of b7-h3, E-cadherin and c-Met in 197 matched tissues of colorectal cancer and adjacent tissues were analyzed by immunohistochemistry. The correlation and clinical significance of the expressions of b7-h3, E-cadherin and c-Met were analyzed by combining pathological data and survival time. 31.5% of the tissue specimens showed normal expression of E-cadherin in the cell membrane, while 68.5% of the tissue specimens showed abnormal expression of E-cadherin in the cytoplasm or not; (2) 71.1% of the tissue specimens showed abnormal expression of c-met, 28.9% of the tissue specimens did not express c-met; (3) further statistics showed that abnormal expression of b7-h 3 and E-cadherin in the colorectal cancer tissue were found. A negative correlation, and the overall survival time of B7-H3 and high expression of E-cadherin in plasma of patients with disease-free survival time decreased significantly; (4) there is a positive correlation between the abnormal expression of B7-H3 and c-Met, and the difference was statistically significant. [Conclusion] in colorectal tumor tissue expression B7-H3, E-cadherin and c-Met pulp And there was a negative correlation between B7-H3 and membrane E-cadherin was positively correlated with c-Met, significantly affect the overall survival time and disease-free survival time. All these results indicate that B7-H3 interacts with c-met can be involved in the regulation of colorectal cancer EMT. The fourth part of B7-H3 via c-met pathway on colorectal cancer cell regulatory mechanisms of EMT [Objective] through To explore the mechanism in colorectal cancer cells in EMT B7-H3 and c-Met interaction, and provide a theoretical basis for the B7-H3 to promote the progression and metastasis of human colorectal cancer. [Methods] the expression of colorectal cancer cell line SW480 B7-H3 as the research object, by CO immunoprecipitation and Westernblot method to observe the B7-H3 and C-M rectal cancer cells The interaction of ET; B7-H3 and c-Met observe the co localization of node of colorectal carcinoma cells by confocal microscopy. The low expression of RKO on colorectal cancer cell B7-H3 as the research object, B7-H3 overexpression and silencing of c-met after incubated by migration assay and scratch test analysis of regulation effect of b7-h3/c-met on invasion and migration of tumor cells, Through dry gene detection and cell ball experiment to explore the effects of b7-h3/c-met on tumor cell stemness. On this basis, exogenous recombinant human B7-H3 protein was observed in c-Met, the expression of p-met; further observed by Westernblot B7-H3 after silencing the expression of c-Met in colorectal cancer cells downstream of key signaling molecule Akt phosphorylation of ERK in water Changes and expression of EMT transcription factor Snail. [results] (1) the over expression of B7-H3 in the SW480 cell stimulatory molecule B7-H3 can be associated with c-Met, by confocal microscopy can be observed between the two co localization; (2) compared with control group, B7-H3 overexpression group of tumor cell surface marker expression dry increased obviously, and the migration, Wound healing and cell sphere formation ability is significantly improved; and compared with B7-H3 overexpression group, B7-H3 overexpression of /c-met silencing tumor cell surface marker dry treated group was significantly reduced, while migration, wound healing and cell sphere formation ability also significantly reduced; (3) human recombinant protein B7-H3 exogenous could increase p-met table Reached the level; (4) when B7-H3 knockdown affects key signaling molecules downstream of Akt, ERK phosphorylation and EMT expression of key transcription factor Snail. [Conclusion] the costimulatory molecule B7-H3 interacts with c-Met, followed by the expression of p-met, and then through the downstream signal axis akt/snail is involved in the regulation of colorectal cancer cells emt, Which affect the metastasis of tumor. In summary, this topic through the analysis of clinical specimens and in vivo and in many aspects and different levels of experiment
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.34

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