【作者】 丁轶；

【导师】 陈龙华；

【作者基本信息】 南方医科大学 ， 放射肿瘤学， 2008， 博士

【摘要】 研究背景和目的原发性肝细胞癌（Hepatocellular carcinoma,HCC）简称肝癌,是一种严重影响人类健康的恶性肿瘤,在我国每年肝癌的发病率为20.37/10万,其死亡率高居癌症死亡率的第二位。目前,临床上可用于治疗肝癌的手段虽然比较多,包括手术、肝移植、血管介入、消融技术、放化疗等,但临床上有一半以上的肝癌患者在行根治性手术前已有了无法检测出的微小转移,它是肝癌术后转移和复发的直接原因。因此,预测肿瘤转移倾向、判断肿瘤预后的标志物,寻找预防和治疗的有效途径,提高病人的5年生存率,降低死亡率是肝癌研究的重点内容,也是肿瘤防治研究中的一个重点和难点。Tiaml（T lymphoma invasion and metastasis 1,T淋巴瘤侵袭转移诱导因子1）基因是鸟苷酸转换因子（guanine nucleotide exchange factors,GEFs）家族成员。GEFs的主要功能是调节Rho GTPase的活性。Rho GTPase家族包括Rho、Rac及Cdc42,是Ras超家族的成员。Tiam1基因体内外均可以特异性地激活RhoGTPase家族中的Rac1。新近的研究发现Tiam1不但激活Rac,更重要的是Tiam1可直接与胞浆和胞膜上的蛋白质相互作用,将它们耦合到Tiam1-Rac信号通路上,从而影响Rac信号通路的特异性。Tiam1基因最初在小鼠T淋巴瘤细胞高侵袭变异株中分离鉴定。随后,在乳腺癌、肺癌及Ras诱导的皮肤癌等肿瘤中证实Tiam1具有明显的促进肿瘤演进和转移的作用。在Tiam1敲除的动物模型中,小鼠皮肤癌发生率明显减低并且肿瘤演进缓慢,Malliri等认为Tiam1在Ras诱导皮肤癌发生的启动和进展阶段发挥关键作用,这一效应与Tiam1表达量有明显的关系。上述的研究表明,Tiam1是多种肿瘤的促癌基因和促进转移基因。本研究拟应用自行设计的包括部分常见恶性肿瘤组织和正常组织的组织芯片,采用免疫组化方法检测和观察人体正常组织和常见恶性肿瘤组织,152例肝癌组织和8种人肝癌细胞株及1种人正常肝细胞株Tiam1蛋白的表达情况;分析Tiam1蛋白在肝癌中的表达及其与各临床病理参数之间的关系,同时结合临床随访资料回顾性分析Tiam1蛋白表达与肝癌患者生存期的关系,寻找提高肝癌预后评价敏感性和特异性的方法;利用基因沉默技术初步研究Tiam1基因在肝癌增殖、侵袭中的作用,探讨Tiam1作为肝癌分子靶点的可行性;为肝癌患者的诊断、预后评价、综合治疗方案设计提供有力的理论依据。方法1、Tiam1蛋白在人体正常组织和常见恶性肿瘤组织的分布和表达研究自行设计并制作了包括鼻咽癌、肺鳞癌、肺腺癌、食道鳞癌、食道腺癌、胃癌、结肠癌、直肠癌、肝癌、胰腺癌、乳腺癌、肾透明细胞癌、宫颈癌、卵巢癌、前列腺癌、膀胱移行上皮癌、脑胶质瘤、皮肤鳞状细胞癌、弥漫性大B细胞淋巴瘤等19种恶性肿瘤及鼻咽、肺、食道、胃、结肠、直肠、纤维平滑肌、肝、胰腺、乳腺、膀胱、子宫颈、卵巢、肾、前列腺、淋巴结、皮肤、脑等18种正常组织的组织芯片,结合免疫组化方法检测Tiam1蛋白在人体正常组织和常见恶性肿瘤组织的分布和表达。2、Tiam1基因/蛋白在人正常肝细胞和肝癌细胞中的表达研究应用荧光定量PCR、免疫细胞化学、Western blot、细胞免疫荧光等方法检测HepG2、Hep3B、SMMC-7721、QGy-7701、QGy-7703、BEL-7402、BEL-7404、BEL-7405等8种肝癌细胞株和人正常肝细胞株HL-7702中的Tiam1基因/蛋白的表达情况。3、Tiam1蛋白在肝癌组织中的表达及临床意义应用免疫组化EnVision^TM二步法检测Tiam1蛋白在152例肝癌组织中的表达情况;应用SPSS13.0软件进行统计学处理,采用x2检验分析Tiam1蛋白表达水平与各临床病理参数之间的关系;采用Kaplan-Meier法回顾性分析Tiam1蛋白表达与肝癌患者生存期的关系;以COX回归对各项指标单因素或多变量联合与生存期的关系进行统计学分析。4、Tiam1基因表达沉默对肝癌细胞生物学特性的影响利用在线数据库和软件设计Tiam1基因干扰片段,用含Tiam1基因的干扰片段瞬时转染肝癌细胞株QGy-7701细胞,荧光定量PCR和Western blot检测Tiam1基因干扰效率。利用MTT法及流式细胞术检测Tiam1基因沉默后对细胞体外增殖的影响;利用体外侵袭小室进行体外侵袭实验,检测Tiam1基因沉默后对肝癌侵袭能力的影响。结果1、Tiam1蛋白在人体正常组织和常见恶性肿瘤组织中的分布和表达情况Tiam1蛋白在正常组织中的乳腺纤维组织、胰腺、肝组织未见阳性表达,而在鼻咽、肺、食道、胃、结肠、直肠、纤维平滑肌、膀胱、子宫颈、卵巢、肾、前列腺、淋巴结、皮肤、脑等15种正常组织中可见不同程度的Tiam1蛋白阳性表达。其中在前列腺增生组织腺上皮、增生平滑肌组织、肾脏远曲小管上皮细胞、血管壁平滑肌等组织中可见Tiam1蛋白中到高表达。Tiam1蛋白在鼻咽癌、乳腺癌、肺鳞癌、肺腺癌、结肠癌、直肠癌、肝癌、前列腺癌、卵巢癌、弥漫性大B细胞淋巴瘤等10种肿瘤组织中高表达;在食道鳞癌、食道腺癌、胃癌、胰腺癌、肾透明细胞癌、宫颈癌、膀胱移行上皮癌、脑胶质瘤、皮肤鳞状细胞癌等9种肿瘤组织中可见Tiam1蛋白低表达。此外,我们还发现肝癌癌旁肝硬化组织和伴有纤维组织明显增生的肝组织中也可见Tiam1蛋白低到中度表达,但表达强度明显弱于肝癌组织,且范围小于肝癌组织。2、Tiam1基因在肝癌细胞株和正常肝细胞株中的表达情况采用荧光定量PCR法检测8种人肝癌细胞株和1种人正常肝细胞株中Tiam1基因的表达情况。单因素方差分析的结果表明:9种细胞株中Tiam1基因的表达差异具有显著性（F=80.514,P＜0.001）,Tiam1基因在8种肝癌细胞中的表达水平均明显高于正常肝细胞中的表达水平,差异具有统计学意义。3、Tiam1蛋白在肝癌细胞株和正常肝细胞株中的表达情况免疫细胞化学检测结果显示,Tiam1蛋白在正常肝细胞株中不表达,在肝癌细胞株中阳性表达。Western blot及细胞免疫荧光检测结果与免疫细胞化学结果一致。4、Tiam1蛋白在肝癌组织中的表达及临床意义免疫组化检测结果显示Tiam1蛋白在152例肝癌组织中的总阳性表达率为88.1%,而在正常组织中呈阴性表达。肝癌组织中各级表达所占比例分别为:“-”占11.9%（18/152）,“+”占24.3%（37/152）,“++”占39.5%（60/152）,“+++”占24.3%（37/152）。Tiam1蛋白表达与肝癌患者的性别、年龄、肿瘤大小、分化程度、肝硬化、转移、复发、血清HBsAg、血清AFP均无相关性（P＞0.05）。随访发现,Tiam1蛋白高表达肝癌患者中位生存时间为27.0个月,低于Tiam1蛋白低表达患者中位生存时间60.0个月,Tiam1高表达患者与低表达患者生存时间有显著性差异（P=0.008＜0.05）。将Tiam1蛋白表达和临床病理参数综合与肝癌患者的生存时间进行单因素分析,结果显示Tiam1蛋白高表达、肿瘤大小、转移、复发和血清AFP值是影响肝癌患者生存时间的重要因素（P值分别为:0.009、0.002、0.030、0.035、0.002）。多因素分析,发现Tiam1蛋白高表达、肿瘤大小和血清AFP值分别是影响肝癌生存预后的独立因素（P值分别为:0.042、0.008、0.001）。5、Tiam1基因表达沉默对肝癌细胞生物学特性的影响利用软件设计合成Tiam1基因的特异性干扰片段,瞬时转染QGy-7701细胞后,荧光显微镜测定转染效率为60%～70%。荧光定量PCR和Western blot结果显示Tiam1基因干扰效率达72%（命名为QGy-7701/Tiam1^-细胞）。MTT法观察Tiam1基因表达沉默后体外细胞的增殖情况,与QGy-7701细胞相比,QGy-7701/Tiam1^-细胞的增殖速度明显减慢,并且呈时间依赖关系（F=237.790,P＜0.001）。利用流式细胞术检测两组细胞的细胞周期结果显示QGy-7701/Tiam1^-细胞周期中S期的比例明显减少（t=14.177,P＜0.001）,表明Tiam1基因表达沉默后细胞增殖缓慢。结果提示Tiam1基因表达水平减低后,肿瘤细胞体外生长被显著抑制。体外侵袭小室实验检测Tiam1基因表达沉默后细胞侵袭能力的改变,结果显示,与QGy-7701细胞相比,QGy-7701/Tiam1^-细胞的侵袭能力明显降低（t=18.054,P＜0.001）,说明Tiam1基因的沉默抑制了肝癌细胞的侵袭能力。结论1.Tiam1蛋白在多种肿瘤组织中高表达,表明Tiam1可能是导致肿瘤发生发展的重要分子;2.Tiam1蛋白在肝癌组织中高表达,在肝硬化组织中弱表达,而在正常肝组织中不表达,提示Tiam1可作为临床肝癌检测的有重要参考价值的标志物;3.Tiam1蛋白高表达结合临床随访资料分析,表明Tiam1与患者预后密切相关,提示Tiam1可作为肝癌预后判断的重要指标,对判断肝癌预后有重要意义;4.Tiam1基因沉默后可抑制肝癌细胞的增殖和侵袭,表明Tiam1基因可促进肝癌细胞的增殖和侵袭,提示Tiam1基因在肝癌细胞的增殖和侵袭中起重要作用。本研究的创新之处1.发现Tiam1与肝癌的增殖、侵袭及预后相关,为开展肝癌基因靶向治疗奠定了理论基础;2.建立了Tiam1基因沉默的肝癌细胞株,为研究Tiam1基因在肝癌发生发展中的作用提供了有价值的研究工具。更多还原

【Abstract】 Background and Objection:Primary hepatocellular carcinoma（HCC） is one of the most forms of malignancy which seriously affect the body health of human being.In our country HCC has the incidence rate of 20.37/10 ten thousand and the second mortality rate in the total cancer mortality.Though many methods including operation,liver transplantation, blood intervention,ablation technique,chemotherapy and radiotherapy,can be performed in HCC therapy,clinically more than one half of HCC patients had the micrometastasis failing to detect before radical surgery,which may be the direct reason of metastasis or recurrence of HCC after surgery.Thus,to predict HCC metastasis tendency,identify the prognosis markers,find the effective way for prevention and therapy and raise five-year survival rate of HCC patients have been the hot spots for HCC studies,and also been an emphasis and difficult point in treatment and prevention of tumor.Tiam1 is one member of guanine nucleotide exchange factors（GEFs）,which mainly act to regulate the Rho GTPase activity.Rho GTPase family is members of Ras superfamily including Rho,Rac and Cdc42.Tiam1 could specifically activate Racl in vivo and vitro,and beside this,recent studies found Tiam1 directly interacted cytoplastic and epimembranal proteins,and then coupled the complex into Tiam1-Rac signaling pathway,thus affecting the specificity of Rac signaling pathway. Tiam1 was initially separated and identified from the variant cell line of mouse T lymphoma with high invasive ability.After that,related studies on breast cancer,lung cancer and skin cancer induced by Ras confirmed that Tiam1 could accelerate tumor progression and metastasis significantly.In the Tiam1-knockout animal model,the incidence rate of mouse skin cancer obviously reduced and tumors were grown slowly. Malliri et al suggested that Tiam1 played the vital role in the initial and progressive stages of induced carcinogenesis of skin,which had the obvious relationship with the amount of Tiam1 expression.The above studies showed that Tiam1 might accelerate carcinogenesis and metastasis in many tumors.In our study,by using tissue chip of normal tissues and common malignant tumors which was designed and produced by ourselves and immunohistochemistry, Tiam1 protein expressions were detected in human normal tissues and common malignant tumors,152 cases of hepatocellular carcinoma tissues,8 human hepatocellular carcinoma cell lines and 1 human normal hepatic cell line.Then the relationship between Tiam1 expressions and various clinopathologic parameters was analyzed,and the relationship between Tiam1 expressions and the life spans of hepatocellular carcinoma patients was also retrospectively analyzed combined with clinical follow-up data in order to find the way to improve the sensitivity and specificity of prognosis evaluation of hepatocellular carcinoma patients.After that, RNA interfere was performed to explore Tiam1 functions in proliferation and invasion of hepatocellular carcinoma.Our study aimed to explore the possibility of Tiam1 as the molecular target of hepatocellular carcinoma and lay the potent theoretical basis on diagnosis,prognosis evaluation and combined therapy design of hepatocellular carcinoma patients.Methods:1.The distributions and expressions of Tiam1 protein in human normal tissues and common malignant tumorsThe tissue chip including 18 normal tissues and 19 common malignant tumors in the whole body was designed and produced by ourselves.Common malignant tumors included nasopharyngeal carcinoma,squamous carcinomas of lung and esophagus, adenocarcinoma of esophagus,gastric carcinoma,colorectal carcinoma, hepatocellular carcinoma,pancreatic carcinoma,clear cell carcinoma of kidney, uterine cervix cancer,ovary carcinoma,prostatic carcinoma,transitional cell carcinoma of bladder,neurogliocytoma,skin squamous carcinoma,diffuse large B lymphoma.Normal tissues included nasopharynx,lung,esophagus,stomach,colon, rectum,fiber and smooth muscle,liver,pancreas,mammary gland,cervix,ovary, kidney,prostate,lymph node,skin and brain.2.The expressions of Tiam1 gene/protein in human normal liver cells and liver cancer cells.Real time PCR,immunocytochemistry,Western blot and cellular immunofluorescence were used to examine the expressions of Tiam1 in 8 hepatocellular carcinoma cell lines named HepG2,Hep3B,SMMC-7721,QGy-7701,QGy-7703, BEL-7402,BEL-7404,BEL-7405 and 1 normal one named HL-7702.3.The expressions of Tiam1 protein in hepatocellular carcinoma tissues and its clinical significance.The expressions of Tiam1 protein in 152 cases of hepatocelluar carcinoma tissues were detected by EnVision^TM two-step immunohistochemistry.The results were analyzed with SPSS13.0 software.The relationship of Tiam1 protein expressions with clinical-pathologic parameters was analyzed with x² test. Kaplan-Meier method was used to retrospectively analyze the relationship of Tiam1 protein expressions with life span of hepatocellular carcinoma patients.The relationship of single factor of each index or multi-variable combination with life span was analyzed by COX regression.4.The effect of Tiam1 gene silencing on the biological characters of hepatocellular carcinoma cells.Tiam1 gene fragments for silencing were designed by online database and software and the fragments were then transiently transfected into QGy-7701 hepatocellular carcinoma cell line.Real time PCR and Western blot were performed to detect the interference efficiency of Tiam1 gene.The effects of Tiam1 gene silencing on cell proliferation in vitro were detected by MTT method and flow cytometry,while those on cell invasion were detected by invasive chamber in vitro.Results:1.The distributions and expressions of Tiam1 protein in human normal tissues and common malignant tumorsNo signals of Tiam1 were observed in fiber and fat of breast,pancreatic gland and liver,while positive signals were observed in 15 normal tissues including nasopharynx,lung,esophagus,stomach,colon,rectum,fiber and smooth muscle, bladder,cervix,ovary,kidney,prostate,lymph node and brain,among which Tiam1 were highly expressed in glandular epithelium of hyperplastic prostate,hyperplastic smooth muscle,distal convoluted tubule of kidney and smooth muscle of vessel wall. Tiam1 proteins were up-regulated in nasopharyngeal cancer,breast cancer,lung squamous cancer,lung adenocarcinoma,colon cancer,rectal cancer,hepatocellular carcinoma,prostate cancer,ovary cancer and diffuse large B cell lymphoma,while low-regulated in adenocarcinoma and squamous carcinoma of esophagus,gastric carcinoma,pancreatic carcinoma,clear cell carcinoma of kidney,cervical carcinoma, transitional cell carcinoma of bladder,glioma and skin squamous carcinoma.In addition,we found that liver sclerotic tissues or liver tissues with obviously hyperplastic fibers besides liver cancer showed Tiam1 expressions at the low or moderate degree.Their expression degree and scope were lower than those in liver cancer.2.The expressions of Tiam1 gene in human normal liver cell line and liver cancer cell linesTiam1 gene expressions in 8 human hepatocellular carcinoma cell lines and 1 human normal hepatic cell line were detected by real time PCR.The results of one-factor analysis of variance showed that the expressions of Tiam1 in 9 cell lines had significant difference（F=80.514,P＜0.001）.The expressions of Tiam1 in 8 hepatocellular carcinoma cell lines were higher than those in normal hepatic cell line with significant difference.3.The expressions of Tiam1 protein in human normal hepatic cell and hepatocellular carcinoma cellsNo signals of Tiam1 expression were observed in normal hepatic cell line while positive signals in 8 hepatocellular carcinoma cell lines by immunocytochecmistry. The above result was consistent with those of Western Blot and cellular immunofluorescence.4.The expressions of Tiam1 protein in hepatocellular carcinoma tissues and their clinical significanceThe total positive expressive rate of Tiam1 protein in 152 cases of hepatocellular carcinoma was 88.1%,but no expression in normal hepatic tissues by immunohistochemistry.The ratio of every grade of hepatocellular carcinoma tissues was respectively that:"-" was 11.9%（18/152）;"+" was 24.3%（37/152）;"++" was 39.5%（60/152）;"+++" was 24.3%（37/152）.Tiam1 expressions had no association with sex,age,tumor size,differentiation,cirrhosis,metastasis,recurrence,serum HBsAg,serum AFP（P＞0.05）.By follow-up analysis,hepatocellular carcinoma patients with high Tiam1 expression had 27.0 months of median survival time,and the survival time had significant difference between patients with high Tiam1 expression and those with low Tiam1 expression（P=0.008＜0.05）.The expressions of Tiam1 protein,tumor size,metastasis,recurrence and serum AFP value were important factors affecting the survival time of hepatocellular carcinoma patients（P value was 0.009,0.002,0.030,0.035,0.002 respectively）.Multi-factor analysis showed that the high expression of Tiam1 protein,tumor size or serum AFP value were independent factors affecting the survival prognosis of hepatocellular carcinoma （P value was 0.042,0.008,0.001 respectively）.5.The effect of Tiam1 gene silencing on the biological characters of hepatocellular carcinoma cellsThe specific interference fragments of Tiam1 gene were produced and transiently transfected into QGy-7701 cell line,with 60%-70%of the transfective efficiency detected by fluorescence microscope.The results of real time PCR and Western blot showed that the interference efficiency of Tiam1 was 72%（named QGy-7701/Tiam1^- cell line）.MTT method was carried out to observe the cell proliferation in vitro after Tiam1 gene silencing.Compared to QGy-7701 cell line,QGy-7701/Tiam1^- cell line had slower proliferative rate in the time-dependent manner（F=237.790,P＜0.001）. The cell cycle of QGy-7701/Tiam1^- was obviously reduced compare with QGy-7701 by flow cytometry（t=14.177,P＜0.001）,indicating that cell proliferation became slowly after Tiam1 gene silencing.The results showed that growth in vitro of tumor cells were obviously inhibited after the expressive level of Tiam1 decreased.The changes of cell invasive abilities after Tiam1 gene silencing were detected by invasive chamber experiment in vitro and the results showed that compared to QGy-7701 cell,the invasive abilities of QGy-7701/Tiam1^- cell decreased obviously （t=18.054,P＜0.001）,suggesting that Tiam1 silencing inhibited the invasive abilities of hepatocellular carcinoma cell.Conclusions:1.Tiam1 protein was highly expressed in many tumors and it can be acted as one important tumor maker for tumorigenesis and development;2.Tiam1 protein was up-regulated in hepatocellular carcinoma tissues,expressed lowly in liver sclerotic tissues and had no expression in normal hepatic tissue, indicating that Tiam1 may be regarded as the important marker for clinical detection of hepatocellular carcinoma;3.Combined evaluation of Tiam1 protein high expression and clinical follow-up data analysis showed that Tiam1 had close relationship with prognosis of hepatocellular carcinoma patients.Tiam1 may act as the important marker for judging prognosis of hepatocellular carcinoma;4.Tiam1 gene silencing can inhibit the proliferation and invasion of hepatocellular carcinoma cell,indicating Tiam1 can promote the proliferation and invasion of hepatocellular carcinoma cell and suggesting the important role of Tiam1 gene in proliferation and invasion of hepatocellular carcinoma cell.Innovations of our study:1.Tiam1 gene had relationship with proliferation,invasion and prognosis of hepatocellular carcinoma,which laid theoretical basis on targeted therapy of hepatocellular carcinoma;2.The establishment of a hepatocellular carcinoma cell line with Tiam1 stable silencing will be provide a valuable tool for Tiam1 functional studies.更多还原