【作者】 刘辉；

【导师】 左连富；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 目的:胃癌是全世界最常见的恶性肿瘤之一,在我国居各类恶性肿瘤之首。近年来的研究表明,胃癌的发生发展是一个多基因参与的多阶段过程。因此,探讨胃癌的发病机制,寻找新的预防和治疗靶点以及有效的治疗药物,对降低胃癌发病率和死亡率,将有十分重要的意义。受体酪氨酸蛋白激酶(Receptor tyrosine kinase, RTK)是外界刺激信息传递至细胞核,转化成细胞效应的信号转导通路中的关键分子组成。EPH(erythropoietin-producing hepatocellular)基因家族是新发现的最大的RTK家族,而EphA2是该亚家族第三个发现并被克隆全长cDNA的成员,是该亚家族成员中被发现有酪氨酸激酶活性的第一个基因。在正常细胞,EphA2严格定位于上皮细胞膜表面的细胞间连接处,可与相邻细胞膜表面的配体EphrinA1结合后形成受体-配体复合物,继而激活胞浆的酪氨酸激酶而活化,参与胚胎发育、细胞迁移导向和血管形成等许多生理过程。EphA2正常表达时启动的信号转导途径抑制了Ras/MAPK的级联反应,削弱PDGF、EGF、VEGF等多种生长因子对MAPK的活化而抑制细胞的生长,并通过破坏细胞的灶状黏附对细胞粘附进行负调节。近年来EphA2在癌症发生发展中的作用越来越受到人们的关注。研究发现EphA2受体在许多肿瘤中有高表达和高活性,包括乳腺癌、大肠癌、前列腺癌及非小细胞肺癌,这些肿瘤细胞的生长、转化、转移与它的突变、过表达、异常定位或(和)异常活化有密切关系。因此,EphA2可能是一个肿瘤临床诊治的新靶点,但目前关于EphA2在胃癌中的异常表达尚缺乏详实的实验依据。格尔德霉素(Geldanamycin, GA)属于苯醌胺莎类抗生素,其作用靶点为热休克蛋白90(Heat Shock Protein, HSP90),为HSP90活性的抑制剂。HSP90是一组高度保守的分子伴侣,广泛存在于生物体中,在肿瘤细胞中异常高表达,其下游底物蛋白参与了肿瘤进展的所有关键环节。HSP90的底物蛋白种类繁多,包括甾体激素受体、苏氨酸激酶受体、酪氨酸激酶受体等,EphA2作为RTK家族一员很可能是HSP90下游的底物蛋白。近年来研究表明,格尔德霉素对多种肿瘤细胞具有明显的抑制作用,并能诱导细胞凋亡,已成为抗肿瘤新药研究的热点。但格尔德霉素对胃癌的抑瘤作用至今未见报道,且其对细胞周期的影响及促凋亡机制是否通过调节EphA2蛋白表达量来实现尚未阐明。RNA干扰(RNA interference, RNAi)是由双链RNA(double-stranded RNAs, dsRNA )分子介导的序列特异性转录后基因沉默( post- transcriptional gene silencing, PTGS)过程,为dsRNA分子在mRNA水平上关闭相关基因表达的过程。目前,RNAi技术在人类基因功能研究和药物靶点鉴定等方面备受青睐,并且具有潜在的临床应用价值。为此,本研究探讨了EphA2及其配体EphrinA1在胃癌发生和发展中的表达情况及其与临床病理特征的关系,分析EphA2表达与胃癌细胞间粘附力、细胞增殖、细胞凋亡及血管形成的关系;观察HSP90抑制剂格尔德霉素对人胃癌细胞株MGC-803增殖和凋亡的影响,并通过检测EphA2、Survivin和Caspase-3下游蛋白表达,深入探讨EphA2是否是GA抗胃癌治疗的作用靶点;并应用RNAi技术沉默EphA2,观察人胃癌细胞株MGC-803的增殖情况,进一步阐明EphA2基因表达与胃癌形成的关系,以期为EphA2成为胃癌临床预防和治疗的新靶点提供理论依据,也为最终阐明胃癌发生和发展的分子机制奠定基础。方法:1 EphA2及其配体EphrinA1在胃癌、癌旁胃黏膜、手术切缘正常胃黏膜中的表达及意义采用免疫组织化学方法检测82例原发性胃癌组织、同个体的癌旁胃粘膜(距癌边缘2-5 cm)和手术切缘正常胃黏膜(距癌边缘5 cm以上,组织学上证实)组织中EphA2、EphrinA1和E-cadherin蛋白的表达,分析其表达与胃癌临床病理特征的关系;采用逆转录聚合酶链反应(RT-PCR)及Western Blot技术对其中随机选取的30例标本进行EphA2 mRNA及其蛋白的定量检测。采用CD34抗体对82例癌组织标记微血管内皮细胞,计算微血管密度(MVD),分析EphA2表达与MVD之间的相关性,及它们与胃癌临床病理特征的关系;采用流式细胞术(FCM)检测82例癌组织中细胞凋亡率及增殖指数(PI),分析EphA2表达与细胞凋亡、增殖的关系;2研究格尔德霉素对人胃癌细胞株MGC-803增殖和凋亡的影响,探讨其调控EphA2表达的作用分子机制。常规培养人胃癌细胞系MGC-803。采用MTT法检测不同浓度的格尔德霉素(20、40、200、400、2000nmol/L)在12h、24h、48h及72h四个时间点对MGC-803细胞的增殖抑制作用;流式细胞术(FCM)测定细胞周期变化及细胞凋亡率;姬姆萨(Giemsa)染色法观察细胞形态学改变;免疫细胞化学染色法及流式细胞术分析EphA2、Survivin及Caspase-3蛋白的表达变化。3 RNAi沉默EphA2基因对人胃癌细胞株MGC-803增殖和凋亡的影响由武汉晶赛公司根据人EphA2基因序列转录RNA的位置及小干扰RNA(small interference RNA, siRNA)设计原则,设计了3个靶位点,并构建了3个针对靶位点的一个载体编码一个小发夹状RNA(small hairpin RNA, shRNA)的质粒表达载体(S1、S2和S3),同时合成了一个阴性对照质粒表达载体(HK)。这些载体能表达绿色荧光蛋白(green fluorescence protein,GFP)和具有新霉素(neomycin,neo)抗性。采用阳离子脂质体将它们转染入人胃癌MGC-803细胞;FCM、激光共聚焦显微镜观察转染效率,选择出阳离子脂质体和质粒表达载体最合适的比例;MTT观察转染效果最佳时间,用于后续实验;在转染后48h,分别采用RT-PCR和Western blot观察MGC-803细胞EphA2 mRNA和蛋白沉默效果,筛选出干扰效果最好的质粒表达载体,用于后续实验。采用干扰效果最好的质粒表达载体转染入MGC-803细胞后,观察EphA2表达、细胞增殖活性、细胞凋亡率及细胞周期的改变。结果:1不同病变胃组织EphA2蛋白及其配体EphrinA1的表达及与胃癌临床病理特征的关系免疫组化结果显示EphA2、EphrinA1蛋白表达强度:胃癌组明显高于癌旁组及正常胃粘膜组(P<0.01);中低分化组明显高于高分化组(P<0.05);深层浸润组明显高于浅层浸润组(P<0.05);淋巴结转移组显著高于无淋巴结转移组(P<0.05)。E-cadherin蛋白表达强度:胃癌组明显低于癌旁组和正常胃粘膜组(P<0.01);中低分化组明显低于高分化组(P<0.05);深层浸润组明显低于浅层浸润组(P<0.05);淋巴结转移组显著低于无淋巴结转移组(P<0.05)。EphA2与EphrinA1两者呈显著正相关(r=0.707, P<0.01);两种蛋白均与E-cadherin呈显著负相关(r=-0.231, r=-0.403, P<0.01)。RT-PCR结果显示EphA2 mRNA在各组中的表达水平无显著性差异(P>0.05),Western Blot与免疫组化结果一致。FCM结果显示EphA2高表达组细胞凋亡率显著低于低表达组(P=0.018),而其增殖指数(PI)显著高于低表达组(P=0.002)。胃癌组织中MVD显著高于癌旁组织及正常胃粘膜(P<0.01);MVD值与胃癌的分化程度、浸润深度及淋巴结转移有关(P<0.05),MVD值还与肿瘤直径有关(P<0.05);EphA2表达与MVD呈显著正相关(r= 0.485, P<0.01)。2格尔德霉素(geldanamycin, GA)对人胃癌细胞株MGC-803增殖和凋亡的影响及调控EphA2等蛋白的表达各浓度组GA均可显著抑制MGC-803细胞的增殖,且呈时间和剂量依赖关系,最大抑制率可达(77.69±0.91)%;GA作用48h后的IC50为558.94nmol/L;各浓度组GA均可阻滞MGC-803细胞于S期,诱导细胞凋亡率的显著升高(P<0.01);姬姆萨染色可见用药组细胞出现凋亡形态学改变:细胞核染色质浓缩、深染,可见核碎裂和凋亡小体;各浓度组GA可显著抑制EphA2、Survivin蛋白的表达,同时上调Caspase-3蛋白的表达(P<0.01)。3 RNAi沉默EphA2基因对人胃癌细胞MGC-803增殖和凋亡的影响当阳离子脂质体/质粒表达载体=2μl/1.0μg转染人胃癌MGC-803细胞时,转染效率能达到86.4%。MTT实验显示:转染后48h EphA2沉默效果最好。RT-PCR和Western Blot显示质粒载体S3对人胃癌MGC803细胞EphA2沉默的效果最好。EphA2表达沉默后抑制MGC-803细胞增殖,在48小时效果最好,随后逐渐下降。EphA2表达沉默后诱导MGC-803细胞凋亡,并可阻滞细胞进程,使G0/G1期细胞比例显著增加。结论:1首次联合检测EphA2、EphrinA1及E-cadherin蛋白在胃癌中的表达,证实EphA2在胃癌组织中异常高表达,并与胃癌浸润深度、分化程度降低及淋巴结转移密切相关。联合检测三种蛋白对胃癌转移、预后及恶性发展的监控具有重要意义。2 EphA2在胃癌组织中异常高表达,可抑制细胞凋亡,促进细胞增殖,在胃癌的发生发展中发挥重要作用,其机制与EphA2 mRNA的转录异常无关,而可能是其翻译水平上调或蛋白质稳定性增高所致。3 EphA2在胃癌组织中的特异性高表达,可能参与肿瘤血管的生成,与胃癌的恶性进展具有密切关系。4首次报道格尔德霉素可抑制胃癌细胞增殖,阻滞细胞周期进程,诱导细胞凋亡,并进一步阐明其机制可能与下调EphA2等蛋白表达有关。5首次应用RNAi技术探讨EphA2表达在胃癌中的作用:EphA2基因沉默后,可抑制胃癌细胞的增殖,诱导其发生凋亡,提示EphA2有望成为预防胃癌发生和治疗胃癌的新靶点。更多还原

【Abstract】 Objectives: Gastric carcinoma, a leader compared with other malignant tumor, is one of the most common malignant tumors. Recently years research shows that the genesis and development of gastric carcinoma is a process involving multistep and multi-gene. Therefore, it is extremely important to investigate pathogenesis and search new target of prevention and therapy and effective drug in gastric carcinoma for decreased morbidity and mortality.Receptor tyrosine kinase (RTK) transmits extracellular signals to nuclear, and then initiates a cascade of signaling events that modulate cellular responses.The EPH (erythropoietin-producing hepatocellular) gene family represents the largest known family of receptor tyrosine kinases. EphA2 was the 3rd found and cloned out the whole long of cDNA and was the first gene with tyrosine kinases activity in EPH family. In normal cells, EphA2 appears to be restricted to intercellular junctions between epithelial cells, where it binds ligands, anchored to the membrane of adjacent cells, to be a complex, which then initiates tyrosine kinases in cytoplasm and participates in embryonic development, cell migration and vasiformation and so on. Normal EphA2 expression signal weaken Ras/MAPK cascade reaction, which activated by PDEF, EGF, VEGF, and restrain cell growth, then negatively regulate cellular adherin through breaking spot-adherin. EphA2 was paid more and more attention in carcinogenesis and development in recent years. It’s found that EphA2 is overexpressed in many human cancers such as breast, colon, prostate and nonsmall-cell lung cancer (NSCL), whose growth, transformation and metastasis correlate with EphA2 mutation, over expression, abnormal location and (or) abnormal inactivation. Above all, EphA2 is a new target for clinical diagnosis and therapy, while there is not exactly experimental basement for EphA2 abormal expression in gastronoma.Geldanamycin (GA), benzoquinoid ansamycin antibiotic, targets heat shock protein 90 (Hsp90), is an inhibitor for Hsp90. Hsp90 is a group of highly conservative molecular chaperones, which is widely found in organism. EphA2, abnormally highly overexpresses in tumor, whose downstream proteins take part in key point of tumor progression. There are a lot of Hsp90 downstream proteins including steroid receptors, therosine kinase receptors and ammonia kinase receptors etc. EphA2, as a member of RTK family, is probably a downstream protein of Hsp90. In recent years’research, GA has become a novel target for anticancer drug development because it is a potent inhibitor of many kinds of tumor and it can induce apoptosis. However, tumor-suppress effect of GA on gastric carcinoma has never been documented, and it is unknown that if GA achieves its way of cellular cycle-modulation and apoptosis-induction through the expression of EphA2 protein.RNA interference (RNAi) is a sequence-specific and post-transcriptional gene silencing process mediated by double-stranded RNAs (dsRNA) through direct mRNA degradation. Currently, RNAi technology is not only a powerful tool to exploit function of human genome and identify target of drug, but also holds great promise in the field of clinical applications.Therefore, our investigation was following: significance of EphA2 and its ligand EphrinA1 in carcinogenesis and progression of gastric carcinoma, and the relationship between these proteins expression in gastric carcinoma; relationship between EphA2 expression and adherin, proliferation, apoptosis and angiogenesis in gastric carcinoma; effect of GA, inhibitor of Hsp90, on proliferation, apoptosis of human gastric carcinoma cells-MGC803; effect of GA on expression of EphA2, Survivin and Caspase-3 protein, further investigation for whether EphA2 was a target of GA on human gastric carcinoma therapy; silencing EphA2 expression by RNAi, and then testing proliferation on human gastric carcinoma cell line MGC-803 contributed to explaining the relation between EphA2 expression and carcinogenesis in gastric carcinoma. It may provide theoretical evidence for whether EphA2 is a new target for preventing and treating gastric carcinoma. It may establish fundament for highlighting mechanism of carcinogenesis and progression of gastric carcinoma as well.Methods:1 Studies on EphA2 and its ligand EphrinA1 expression in gastric carcinoma, paired adjacent mucosa to gastric carcinoma and normal gastric mucosa at surgical margin of gastric carcinomaEphA2, EphrinA and E-cadherin proteins expression were detected by means of immunohisochemistory (IHC) in 82 cases surgical resected primary gastric carcinoma tissues, paired adjacent mucosa (2~5 cm from margin of gastric carcinoma ) and normal mucosa at surgical margin (at least 5cm from margin of gastric carcinoma and histologically proven) tissues, and then analyzed the relation between their expression and clinic charactors; EphA2 mRNA and protein expression were detected in 30 cases drawn from above 82 cases randomly by RT-PCR and Western Blot.Neovasculature was detected using immunohistochemistry labeled with CD34 antibody and indicated by microvessel density (MVD) in 82 tissues of gastric carcinoma, analyzing the correlation between EphA2 and MVD, between these proteins and clinic characters of gastric carcinoma. Apoptosis rate and proliferative index (PI) were detected in 82 cases by FCM, and then the relation between EphA2 expression and apoptosis, proliferation was analyzed.2 Effect of GA on proliferation and apoptosis of human gastric carcinoma cells-MGC803 and its regulation mechanism to EphA2Human gastric carcinoma cell line MGC-803 was conventional cultured. Effect of GA (20, 40, 200, 400, 2000 nmol/L) for 12h, 24h, 48h and 72h on proliferation suppressions of MGC-803 cells were detected by MTT assay; cell-cycle change and apoptosis rate were detected by FCM; cell morphology change was observed by Giemsa staining; expression of EphA2, Survivin and Caspase-3 protein detected by IHC and FCM.3 Effect of RNAi silencing EphA2 on proliferation and apoptosis of human gastric carcinoma cell line MGC-803Three locations targeted were designed and three plasmid vectors (S1, S2 and S3) expressed shRNAs for silencing EphA2 were constructed according to transcription RNA location of human of EphA2 gene sequence and designing principle of small interference RNA (siRNA) by Wuhan JingSai Company. Also a negative control plasmid vector HK was constructed. These plasmid vectors can express green fluorescence protein (GFP) and resist neomycin (neo). They were transfected into human gastric carcinoma MGC-803 cells by cations liposome vector. The transfection efficiency of plasmid vector was evaluated by calculating the ratio of fluorescent cells to total cells tested by FCM and Laser confocol scan microscope to select the most suitable ratio of cations liposome vector to plasmid vector; the best transfection time was selected by MTT and used in later experiment; EphA2 mRNA and protein of MGC-803 cells were detected by RT-PCR and Western blot 48h after transfection, respectively, and the best plasmid vector silencing EphA2 of MGC-803 cells was selected and used in later experiment; Changes of EphA2 protein, proliferative activity, cell cycle and apoptosis were detected in MGC-803 cells transfected with the best plasmid vector silencing EphA2.Results:1 EphA2 and its ligand EphrinA1 protein expression in various lesions of gastric tissues and relationship between clinic charactersEphA2 and EphrinA1 protein expression rates shown by IHC: higher in carcinoma than in adjacent group and normal group (P<0.01); higher in medium and low differentiation than in well differentiation (P<0.05); higher in deeper infiltration than in superficial layer (P<0.05); higher in lymph node metastasis than in non-lymph node metastasis (P<0.05). E-cadherin expression rates: lower in carcinoma than in adjacent group and normal group (P<0.01); lower in medium and low differentiation than in well differentiation (P<0.05); lower in deeper infiltration than in superficial layer (P<0.05); lower in lymph node metastasis than in non-lymph node metastasis (P<0.05). EphA2 expression was positively correlated with EphrinA1 significantly (r=0.707, P<0.01); above two protein were negatively correlated with E-cadherin significantly (r=-0.231, r=-0.403, P<0.01). There is no difference of EphA2 mRNA among every group shown by RT-PCR (P>0.05), while Western Blot results were identical with IHC. Apoptosis rate was higher in EphA2 high-expression group than in low-expression (P=0.018) and PI was contrary (P=0.002) shown by FCM.MVD was significantly higher in gastric carcinoma than in adjacent and normal tissues (P<0.01); MVD was correlation with differentiation, infiltration depth, lymph node metastasis and diameter of tumor (P<0.05); EphA2 protein expression was positively related with MVD significantly (r= 0.485, P<0.01).2 Proliferative and apoptotic effect of GA on human gastric carcinoma cell line-MGC803 and regulation of GA on EphA2 and other proteinsEvery group of GA inhibited proliferation of MGC-803 cells significantly in time-dependent manner and dose-dependent manner and the highest inhibition rate reached (77.69±0.91) %; IC50 was 558.94nmol/L after 48h of GA implication. Every group of GA blocked MGC-803 cell cycle at S-phase and induced apoptosis higher significantly (P<0.01); Giemsa staining showed apoptosis morphologic change after GA: concentration and dark staining of cytoplasm, karyorrhexis and apoptotic bodies; Every group of GA inhibited the expression of EphA2 and Survivin proteins at the same time increased Caspase-3 expression (P<0.01).3 Effect of RNAi silencing EphA2 on proliferation and apoptosis of human gastric carcinoma cell line MGC-803When the ratio of cations liposome vector to plasmid vector was 2μl to 1.0μg, the transfection efficiency was 86.4%. The time was 48h by MTT and the vector was S3 by RT-PCR and Western Blot were the best for EphA2 silence. RNAi silencing EphA2 mRNA and protein in MGC803 cells inhibited proliferation, decreased after 48h, and induced apoptosis, furthermore, increased the ratio of G0/G1-phase significantly.Conclusions:1 It’s the first time that detecting combinativly of EphA2, EphrinA1 and E-cadherin protein expressions in gastricarcinoma and confirming EphA2 abnormal expression in gastricarcinoma, which involved in infiltrative depth, differentiation and limphatic metastasis. Detecting the three protein levels can provide certain reference value in evaluating the metastasis, prognosis and malignant development monitoring of gastric carcinoma.2 Over expression of EphA2 in gastric cancer may inhibit cell apoptosis and promote cell proliferation, and plays an important role in the carcinogenesis of gastric carcinoma, its mechanism may not be related to the level of mRNA, which may result from the up-regulation in translational level or the increased protein stability.3 EphA2 is specifically over expressed in gastric cancer and may be involved in angiogenesis and plays an important role in the initiation and progression in gastric carcinoma.4 We provided the first evidence that GA can inhibit the proliferation, arrest cell cycle and induce the apoptosis of gastric carcinoma MGG-803 cells. The mechanism may be related with down-regulation of EphA2 and other proteins.5 It’s the first time to investigate EphA2 expression in gastricarcinoma by RNAi: silencing EphA2 could inhibited proliferation and induced apoptosis. These suggested that EphA2 may be a new target for preventing gastric carcinogenesis and treating gastric carcinoma.更多还原