【作者】 柯爱武；

【导师】 刘康达；

【作者基本信息】 复旦大学 ， 肿瘤学， 2008， 博士

【摘要】 肝癌是国内常见的恶性肿瘤之一,其侵袭转移与复发是患者死亡的主要原因之一。因此,探索肝癌侵袭转移与复发的发生机制,寻求有效的抗肝癌转移与复发治疗措施,对改善肝癌患者的预后具有重要意义。在肝癌侵袭转移与复发中,HGF/c-Met信号系统的“对话”有着重要地位,这一作用在肝癌切除术后,残留肝脏再生,致HGF/c-Met系统处于活跃的状态,从而促进残留肝癌细胞增殖使其与肝癌术后转移、复发的关系也表现尤为突出。HGF/c-Met信号系统在肝癌侵袭转移与复发中的重要地位,也为肝癌侵袭转移与复发的治疗提供了新的靶点。当前,已发现多种HGF/c-Met信号的抑制剂,但所发现的HGF/c-Met抑制剂都有不同程度局限,如HGF竞争性拮抗剂NK4,c-Met受体的选择性抑制剂PHA-665752只能在一定程度上抑制HGF/c-Met信号,究其原因是HGF/c-Met与其它信号间的交互作用,如HGF/c-Met信号与整合素信号间的交互作用。有研究表明四跨膜蛋白CD151可与c-Met形成一功能复合物,并在HGF/c-Met信号转导以及其与其它信号间的交互作用中扮演重要的角色,可能为抑制HGF/c-Met信号的全新靶点。目前,国内外还没有CD151在肝癌中研究的报道,同时,CD151在HGF/c-Met信号中的作用也缺乏系统研究。本实验首先在肝癌、相应癌旁、正常肝组织以及不同转移潜能肝癌细胞系中检测CD151表达差异;研究其与肝癌发生、发展以及预后的相关性;通过转染与干扰调节CD151表达后,探讨CD151与肝癌细胞侵袭与转移的关系;最后对CD151在HGF/c-Met信号转导中的具体作用进行研究。第一部分四跨膜蛋白CD151在肝癌中的过表达目的:检测肝癌、癌旁和正常肝组织中四跨膜蛋白CD151的mRNA与蛋白表达,分析其与肝癌发生的关系。方法:应用普通RT-PCR,荧光定量PCR与免疫组织化学技术检测CD151在肝癌、相应癌旁、正常肝组织中的表达,并比较三组间CD151的表达差异。结果:CD151在肝癌、相应癌旁和正常肝组织中均有表达,相对表达量分别为3.6±0.9(0.5-8.2),2.7±0.1(0.0-6.5)与1.8±0.8(0.0-3.1),肝癌与相应癌旁组织间差异明显(p＜0.05),CD151在癌旁与正常肝组织的表达也存在差异(p＜0.05)。免疫组织化学显示CD151主要分布于细胞膜,其在肝癌组织中的表达明显高于后两者(p＜0.05)。结论:四跨膜蛋白CD151在肝癌发生中可能有重要作用。第二部分CD151/或c-Met与肝癌临床病理特征及预后的关系目的:研究CD151和c-Met在原发性肝癌中表达,探讨CD151和c-Met与原发性肝癌的关系及临床生物学意义。方法:应用荧光定量PCR检测120例肝癌组织中CD151与c-Met mRNA的表达,组织芯片检测520例肝癌组织中CD151与c-Met蛋白的表达,SSPS11.5统计分析两者与肝癌临床病理特征及预后的关系。结果:CD151蛋白的过表达与肝癌患者的无包膜、高分期与分级、肿瘤细胞低分化、有门脉癌栓及多卫星灶等临床病理因素有关(p＜0.05);而c-Met与有门脉癌栓、肿瘤直径＞0.5cm、多卫星灶、高分期与分级与肿瘤细胞低分化(p＜0.05)等相关。CD151与c-Met在肝癌中表达呈中度相关,有显著性意义(r=0.051,p=0.013),CD151~+组的3、5、7年生存率明显低于CD151组(p＜0.01),CD151~+/c-Met~+组的3、5、7年生存率明显低于CD121~-/c-Met~+及CD151~-/c-Met~-组(p＜0.01)。结论:四跨膜蛋白CD151与原发性肝癌的侵袭、转移及不良预后相关;CD151对c-Met生物学功能可能存在影响。第三部分CD151表达与肝癌细胞侵袭与转移的关系目的:研究四跨膜蛋白CD151与肝细胞癌细胞侵袭与转移的关系。方法:荧光定量PCR,western blot及流式细胞仪检测不同转移潜能肝癌细胞中CD151的表达差异;转染pcDNA3-CD151cDNA与pGPU6/GFP/Neo-CD151,筛选出稳定细胞株,经western blot,流式细胞仪验证CD151表达改变后,明胶酶谱研究基质金属蛋白酶分泌变化;MTT检测CD151表达改变后,细胞增殖能力变化;transwell研究细胞侵袭能力改变。细胞接种于裸鼠体内,研究CD151表达改变后,细胞成瘤与肺转移能力的改变。结果:转染pcDNA3-CD151cDNA与pGPU6/GFP/Neo-CD151于低转移能力的HepG2与高转移的HCCLM3肝癌细胞,筛选的稳定细胞株CD151表达改变明显;明胶酶谱研究显示CD151的表达与MMP9的分泌呈正相关,而对MMP2的影响较小。肝癌细胞增殖受CD151表达水平影响较小(p＞0.05):CD151高表达组细胞的移动与侵袭能力明显增强,肺转移灶明显增加,且以Ⅲ,Ⅳ期为主。结论:四跨膜蛋白CD151与肝癌的侵袭与转移正相关。第四部分CD151对HGF/c-Met信号转导的影响目的:研究CD151与c-Met在肝癌细胞中的关系及其对HGF/c-Met信号的影响。方法:免疫荧光与激光共聚焦研究两者在不同转移潜能肝癌细胞中的表达与相对分布;免疫共沉淀研究两者形成复合物情况。以HGF作用于CD151表达改变前后的细胞,western blot研究HGF/c-Met下游与肝癌转移相关的重要信号分子Akt、FAK、ERK1/2磷酸化差异,分析其在肝癌细胞中对HGF/c-Met信号的影响。结果:CD151与c-Met主要表达于肝癌细胞的细胞膜,分布一致;免疫共沉淀研究显示两者形成复合物。在HGF作用后,下游信号分子Akt与FAK在CD151表达不同的细胞,磷酸化差异明显(p＜0.05),而ERK1/2的磷酸化在两组间无差异(p＞0.05)。结论:四跨膜蛋白CD151与c-Met在肝癌细胞中形成复合物,其通过Akt与FAK对HGF/c-Met产生影响。更多还原

【Abstract】 Hepatocellular carcinoma(HCC) is one of the most common cancers in China in terms of number of cases,and the invasion and metastasis are the leading cause for the HCC death,so it is of great clinical importance to investigate the mechanism of invasion and metastasis,and the effectual therapeutic way of anti-metastasis and relapse of HCC.Previous study has showed that the HGF/c-Met signal pathway play a crucial role in the invasion and metastasis of HCC,and the more activeness of HGF/c-Met system after hepatectomy due to the regeneration of remnant thepatic tissue,which promotes proliferation of remnant HCC cells.The critical role of HGF/c-Met signaling in HCC has sparked the active interest in searching for the means of the therapy of HCC through targeting it,as a result,many molecules of inhibitor targeting this signaling were come forth during the latest few years,such as the competed and selected inhibitor NK4 and PHA-665752,however,little progression has been achieved due to the cross-talking between the HGF/c-Met signaling pathway and the other signaling pathway,for example the cross-talking between HGF/c-Met and integrin signaling pathway.The latest study had reported the tetraspanin CD151 is involved in the signaling through form a complex with c-Met, but the role and expression of CD151 in HCC,and the detailed mechanism of CD151 in HGF/c-Met signaling pathway remains unknown.In the present study,we investigated the expression of CD151 and c-Met in HCC cell lines with different metastatic potential,and HCC,their adjacent nontumorous and normal liver tissues, and then explored the changes of invasive and metastatic ability in non-metastatic cell line HepG2 through CD151 cDNA transfection and high metastatic cell line HCCLM3 through silencing CD151 in vivo and vitro.We assayed the clinical significance of CD151 and combined expression of CD151 and c-Met in HCC samples using tissue microarrays(TMAs) and quantitative reverse transcription-polymerase chain reaction(qRT-PCR).Finally,the effect of CD151 on the HGF/c-Met signaling pathway was studied. Part one Overexpression of tetraspanin CD151 in hepatocellular carcinomaObjective:To investigate the relationship between the expression of CD151 and the carcinogenesis of HCC.Methods:The expression of CD151 was evaluated in HCC,their adjacent nontumorous and normal liver tissues by qRT-PCR and immunohistochemistry.Result:CD151 mRNA can be detected in all samples of three different type tissues, the CD151 mRNA level was significantly higher in HCC than those in their adjacent nontumorous and normal liver tissues(p＜0.05),the mRNA level in adjacent nontumorous also higher than that in normal liver tissues(p＜0.05).The CD 151 protein was located in the cytoplasm of HCC cell,the level of CD151 protein was consistent with the CD151 mRNA(p＜0.05).Conclusion:The overexpression of CD151 may be related to the carcinogenesis of HCC.Part two Correlation and clinical significance of overexpression of CD151 and/or c-Met in hepatocellular carcinomaObjective:To investigate the correlation and clinical significance of CD151 and/or c-Met with hepatocellular carcinoma.Methods:The qRT-PCR was used to detect the expression of CD151 and c-Met in HCC,the TMA was used to detect the expression of CD151 and c-Met in 520 HCC patients,then the clinical significance of overexpression of CD 151 and/or c-Met was analyzed by SPSS11.5.Result:Overexpresvion CD151 and c-Met accounted of 59.8%(311/520) and 54.4% (283/520) of total patients,respectively.Overexpression CD151 was found to correlate significantly with portal vein tumor thrombus(PVTT),multiple tumor number,high TNM stage and low differentiation(p＜0.05).However,other clinical characteristics,including age,gender,preoperative serum AFP and tumor size were not directly related to the overexpression of CD151.As for the overexpression of c-Met was related significantly to PVTT,large tumor size,multiple tumor number, high TNM stage and low cell differentiation(/7＜0.05).The 3,5,7,year survival rate of CD151~+ was much lower than that of CD151~-(p＜0.01),furthermore,3,5,7 year survival rate of CD151~+/c-Met~+ was much lower than those of CD151~-/c-Met~+ and CD151~-/c-Met~-.Conclusion:The overexpression of CD151 and high expression of CD151/c-Met can be a new marker in predicting the prognosis of HCC,and the tetraspanin CD151 could affect the signaling of HGF/c-Met.Part three Relationship of overexpression CD151 with the invasion and metastasis of hepatocellular carcinomaObjective:To investgate the relationship of CD151 with the invasion and metastasis of HCC cells.Method:the qRT-PCR,western blot and FCM was used to detect the expression of CD151 and c-Met in different HCC cell lines with different different metastatic potential.We then transfected the pcDNA3-CD151 cDNA and pGPU6/GFP/Neo-CD151 plasmid into the non-metastatic cell line HepG2 and the highly metastatic cell line HCCLM3,the expression of CD151 was determined by FCM,western blot in the stable transfection HCC cell lines.The secretion of MMPs was detected by gelatin zymography,and the ability of proliferation and invasion was tested by MTT and transwell in the the stable transfection HCC cell and their parental cell lines,respectively.At the last,metastasis assays in vivo was performed.Result:The secretion of MMP9 was upregulated in CD151 high expression group, while the activity of MMP-2 was not affected by the expression of CD151;the ability of proliferation did not alter between the high and low CD 151 expression groups,the transwell showed that the numbers of invaded cells in HCCLM3,HCCLM3-Mock and HepG2-CD151 were 36.8±14.4,32.3±10.2 and 26±8.8,which were significantly higher than those in HCCLM3-CD151-shRNA and HepG2, HepG2-Mock(20.3±10.7 and 17±7.7,14±6.3).The pulmonary metastasis rates and metastatic tumor clusters per mouse were 100%(5/5) and 220±53,100%(5/5) and 238±55,60%(3/5) and 145±43 in HCCLM3,HCCLM3-Mock cells and HepG2-CD151 cells group respectively,while 40%(2/5) and 114±46,0%(0/5) and 0%(0/5) in HCCLM3-CD151-shRNA cells,HepG2 and HepG2-Mock cells groups, respectively,with a statistical significance(p＜0.05).Conclusion:The CD151 was positive correlation with the ability of invasion and metastasis. Part four Role of CD151 on the HGF/c-Met signaling transduction pathway in Hepatocellular carcinoma cellsObjective:To expatiate the relationship between CD151 and c-Met in HCC cells, and the role of CD151 on HGF/c-Met signal pathway in hepatocellular carcinoma (HCC) cells.Methods:we investigated the expression of CD151 and c-Met in different metastatic potential of HCC cell lines by immunofluorescence and confocal laser scan microscopy,and the presence of CD151/c-Met complex was confirmed in HCCLM3 cells by immunoprecipitation,then the phosphorylation and non-phosphorylation of Akt,FAK and ERK,which are key molecules in HGF/c-Met signal pathway,were tested by western blot after HGF acting on the modified and parental cells.Result:The expression and location of CD151 and c-Met were unanimous,and formed a complex in HCC cells.The phosphorylation of FAK and Akt was higher in high expression of CD151 cells than in low expression cells(p＜0.05),while the phosphorylation of ERK had no difference between the two kinds of HCC cells (p＞0.05).Conelusion:The CD151 affect the HGF/c-Met signal transduction pathway through the signal molecules of FAK and Akt.更多还原