【作者】 刘健；

【导师】 沈锋；

【作者基本信息】 第二军医大学 ， 肝胆外科学， 2010， 博士

【摘要】 肝细胞癌(HCC)是人类最常见的致命性肿瘤之一,在全球肿瘤死亡原因中排列第五,中国大陆肝癌发病率占全球发病率的55%。根治性切除是HCC治疗的首选治疗方法,但是预后尚不理想,首要原因是肝内复发。寻找理想的肝癌术后预后价值指标,并据此选择合理的辅助治疗对改善预后有重要意义。目前临床上对肝癌预后的判断主要依靠临床病理学,如TNM分期,但是它仅能对中晚期肝癌较好的做出预后判断,对于病理特征较相似的早期肝癌则在应用上有缺陷。近年研究表明,早期肝癌尽管有相似的临床病理特征,但根治性切除后病人的复发和生存情况却迥异,较大部分患者术后也会出现早期复发,据报道五年复发率近60%；按当前肝癌治疗方法的共识(如Barcelona Clinic Liver Cancer)接受肝切除者多为早期肝癌,并且随着高危人群防癌意识和早诊水平的提高,临床上早期肝癌接受肝切除者日益增加,故寻找对早期肝癌预后判断的分子标记甚为重要,迄今为止应用临床的肝癌术后预后分子标志,尤其是适用于早期者仍然缺乏。因此探索与肝癌转移和复发相关的分子,研究其调节肝癌细胞增殖和侵袭的机制,并按照该分子区分患者的预后,具有重要的临床意义。本课题在我们既往以基因芯片筛选出肝癌组织中AAH特异性高表达和体外能够调节肝癌细胞增殖、侵袭的基础上,研究AAH的调节机制以及其表达水平对肝癌术后预后尤其是探索其对早期肝癌预后的是否有判断作用,为使其成为肝癌治疗靶点提供理论和临床依据。第一部分AAH(天冬氨酸β羟化酶)在肝癌中的表达和临床意义目的在前期研究AAH在肝癌组织中特异性高表达和体外能够调节肝癌细胞增殖、侵袭的基础上,研究AAH的表达水平对肝癌术后预后的影响,尤其是对早期肝癌预后的预测。方法cDNA芯片研究配对肝癌组织和癌旁组织中基因表达情况,并以RT-PCR和免疫组化证实cDNA芯片的结果。前瞻性观察2004.1-2004.6中连续入组的233例接受根治性切除患者癌组织中AAH的表达水平和预后的关系,特别是对BCLC早期(O/A)患者的预后。结果cDNA芯片检测肝癌基因表达谱发现AAH在肝癌组织中的表达比癌旁组织高12.5倍,RT-PCR和免疫组化分别从mRNA和蛋白水平证实AAH在肝癌组织中呈过表达。分析发现AAH的表达水平与肿瘤患者血清AFP水平、肿瘤直径、肿瘤数目和TNM分期有关；AAH高表达患者复发率显著高于低表达患者,生存率则相反；多因素分析AAH是影响患者复发和生存的独立因素(hazard ratio HR,95%confidence interval, CI,复发：3.161,2.115-4.724, P<0.001;生存：2.712,1.734-4.241,P<0.001)。在BCLC A期患者中,AAH高表达患者复发率高于低表达患者,生存率相反,这在]3CLC A期直径≤5cm和>5cm患者中也得到类似的结果。而AAH的表达水平对BCLC B和C期患者术后预后没有统计学差异。小结肝癌组织中AAH的表达水平与肝癌患者术后预后有关,对BCLC早期肝癌患者预后有预测作用,AAH高表达患者术后可能会出现复发和转移。第二部分Gas6/Ax1信号通路与AAH的关系及其功能目的研究AAH调节肝癌增殖和侵袭的部分机制。方法检测Gas6/Ax1在肝癌组织和肝癌细胞系SMMC-7721、HepG2、Hep3B、PL、Hu7和正常肝细胞系L02中的表达情况；调节AAH在肝癌细胞系SMMC-7721中的表达,western blot检测下游分子Axl活性形式的表达变化以及活性形式的Axl表达变化时,肝癌细胞增殖和侵袭能力的改变,最后以免疫共沉淀证明AAH参与了Gas6/Ax1的相互作用。结果Axl在癌组织和肝癌细胞系中的表达水平高于癌旁组织和正常肝细胞系,而Gas6表达则无差异。调节AAH在肝癌细胞系SMMC-7721中表达时,活性形式的Axl的表达出现同向变化。体外上调Axl表达时,肝癌细胞系SMMC-7721的增殖加快、侵袭能力提高；下调Axl表达时出现相反的变化。免疫共沉淀证明AAH参与了Gas6和Axl相互作用。小结AAH调节肝癌增殖和侵袭可能部分通过Gas6/Ax1信号通路实现,Axl受体在肝癌中起癌基因作用。第三部分Notch1与AAH的关系及其在早期肝癌术后预后中的作用目的Notch家族的配体和受体都有大量的EGF样结构,本部分证明Notch1也受AAH的调节。鉴于Notch1信号通路的功能以及在肿瘤发生中作用,我们推测Notch1在肝癌中的表达变化在肿瘤发生中很早就表现出来,对早期肝癌术后预后有预测作用。方法检测Notch1-4受体在肝癌组织和肝癌细胞系SMMC-7721、HepG2、Hep3B、PLC、Hu7中的表达情况；调节AAH在肝癌细胞系Hu7中的表达,westernblot检测Notch1的表达变化。前瞻性选取2004.1-2004.7年246例根治性切除的肝癌患者为研究对象,以RT-PCR,实时定量RT-PCR、western blot和免疫组化检测Notch1在肝癌组织和癌旁组织中的表达情况,分析其表达水平对直径=5cm和BCLC 0/A期患者的预后的预测作用,并以回顾性研究本院1996-2001年随机选取的227例患者的Notch1的表达水平与预后的关系验证前瞻性研究的结果。结果除Notch1在肝癌细胞系Hu7中表达外，Notch1、2在肝癌组织和肝癌细胞系中都不表达,Notch3和Notch4在肝癌中表达不规则。调节AAH在肝癌细胞系Hu7中表达时,Notch1的表达出现同向变化。Notch1在肝癌组织中的表达低于配对癌旁组织。在前瞻性队列直径=5cm组中,Notch1低表达患者肿瘤复发时间比高表达患者短,患者生存时间相反。多因素分析发现Notch1的表达水平是影响肝癌复发和生存的独立因素。回顾性队列中的结果也证实了这一点。两个队列中还发现Notch1的表达水平对BCLC 0/A期患者的预后也是一个独立影响因素。小结Notchl也受AAH调节,在肝癌中可能发挥抑癌基因作用,其表达水平与早期肝癌患者根治性切除术后预后密切相关。全文结论：AAH在肝癌组织中高表达,能够调节肝癌细胞的增殖和侵袭,并与肝癌患者根治性切除术后预后相关。AAH能够调节癌基因Axl受体的表达,其作用部分机制可能通过Gas6/Ax1信号通路。Notch1也受AAH调节,由于其在肝癌中可能发挥抑癌基因作用,故AAH不通过Notch1通路调节肝癌的增殖和侵袭,但是其表达水平与早期肝癌患者根治性切除术后预后密切相关。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is one of the most prevalent cancers and the 5th leading cause of cancer-related deaths worldwide. Although radical hepatectomy is considered as one of the first line treatment for patients with early HCC, the surgical outcome is far from satisfactory due to the likelihood of intrahepatic and extrahepatic recurrence. In clinical practice, it is still difficult to predict the prognosis for patients with early HCC due to the similarity in clinicopathologic characteristics. In fact, early-stage HCC also tends to recur and the 5-year recurrence rate was up to 60%. Patients with early-stage HCC are recommended to receive hepatectomy. Meanwhile, the number of the early stage HCC patients who undergo hepatic resection has been gradually increasing as a result of progresses in the surveillance and early detection. Although great efforts have been made for investigating precise prognostic biomarker for early HCC, the optimal candidates with clinical applicability are still lacking and urgently required. Therefore, it is clinically meaningful that metastasis and prognosis related molecule and its functional mechanism were found. In this study, the relationship between expression of AAH and the prognosis of HCC patients undergoing curative resection was evaluated based on the former study that AAH was overexpressed in HCC tissues and could regulate the invasiveness and proliferation of HCC cells in vitro.Part 1 Expression and clinical meaning of AAH in hepatocellular carcinomaPurpose:The association between the overexpression of aspartyl-(asparaginyl)-β-hydroxylase (AAH) and the invasiveness of hepatocellular carcinoma (HCC) in vitro has been previously studied and reported. However, the prognostic value of AAH expression in HCC remains unclear. This study was to investigate the relationship between AAH expression and tumor recurrence and patients’survival.Methods:Overexpression of AAH was found by cDNA, and determined by RT-PCR, immunohistochemistry (IHC). The expression of AAH and prognosis of HCC patients undergoing hepatectomy, especially at BCLC 0/A stage patients, was evaluated in 233 consecutive patients recruited between January 2004 and June 2004 prospectively.Results:We identified AAH as the most overexpressed gene in HCC by cDNA microarray hybridization. A prospective observational study of 233 patients undergoing curative resections indicated that AAH expression was an independent factor affecting the recurrence (HR,3.161,95% CI,2.115-4.724, P<0.001) and survival (HR,2.712,95% CI,1.734-4.241, P< 0.001) by multivariate analysis. Patients with AAH overexpression had a poorer prognosis in contrast with those with AAH underexpression (P<0.001 for both recurrence and survival). In BCLC stage A patients with AAH over-or underexpression, the tumor recurrence and survival rates were also statistically different (45% and 85% vs.16% and 33% in 1-and 3-year cumulative recurrence rates; 73% and 37% vs.90% and 80% in 1-and 3-year survival rates? P<0.001 for both). Furthermore, in stage A patients with tumor=5cm in diameter, the time to recurrence (TTR) was 26.7±1.6 versus 51.9±2.8 months, and the one-and three-year survival rates were 97% and 52% versus 100% and 90% in AAH over-and underexpression patients (P<0.001 for both).Conclusion:Consequently, AAH overexpression in HCC was strongly correlated with worse surgical outcomes and this molecule likely provides a more precise prognostic predictor in early stage HCCs.Part 2 The relationship between Gas6/Ax1 pathway and AAH, and the biological function of Gas6/Ax1.Purpose:To investigate the mechanism of AAH regulating HCC proliferation and metastasisMethods:The expression of Gas6 and Ax1 in HCC tissues and cell lines SMMC-7721, HepG2, Hep3B, PLC, Hu7 and HCC adjacent nontumorous tissues, normal cell line L02 were evaluated. The active form of Axl expression was examined by western blot analysis when AAH was up-or down-regulated in SMMC-7721 cell line. In addition, the proliferation and invasiveness of the cell were also evaluated correspondingly. Finally, whether AAH participated in the interaction of Gas6 and Ax1 was confirmed by immunoprecipitation.Results:The expression of Ax1 in HCC tissues and cell lines were higher than that in nontumorous tissues and L02, but no difference was observed in Gas6 expression. And the active form of Axl expression was positively correlated with the expression of AAH. When Axl was up or down regulated, the proliferation and invasiveness of SMMC-7721 altered correspondingly. In addition, that AAH participated in the interaction of Gas6 and Ax1 was confirmed by immunoprecipitation. Conclusion:The mechanism of AAH regulating HCC proliferation and metastasis might be through the Gas6/Ax1 pathway, and Axl played an oncogene role in HCCPart 3 The relationship between Notchl and AAH, and the prognostic value of Notchl in early stage HCCPurpose:Multiple EFG-like domains were found in the ligands and receptors of Notch family. In this part, that whether Notchl could be regulated by AAH would be demonstrated. Considering the function of Notchl signaling pathway and its role in tumorigenesis, we inferred that abnormal expression of Notchl could be detected in the early process of HCC formation, and could predict the surgical outcomes of early stage HCC patients.Methods:The expression of Notchl-4 in HCC tissues and cell lines SMMC-7721, HepG2, Hep3B, PLC, and Hu7 was evaluated. The expression of Notchl was examined by western blot when AAH was up-of down-regulated in Hu7 cell line. The differential expression of Notchl was also examined by RT-PCR, western blot and immunohistochemistry. The correlation between expression level of Notchl and outcome after curative hepatectomy was investigated in training cohort of 246 patients with hepatocellular carcinoma recruited prospectively, and verified by the results of validation cohorts with 227 patients retrospectively. Patients with tumor =5cm in diameter from two cohorts which were recognized as training set (n=83), validation set (n=108), respectively, and patients at BCLC early (0/A) stage were further investigated.Results:Neither Notchl nor Notch2 could be found in HCC tissues nor cell lines, except that Notchl could only be found in Hu7 cell line. The expression of Notch3 and Notch4 were irregular. Expression of Notchl was positively correlated with the expression of AAH by western bot analysis. Compared with paired nontumorous tissue, tumor tissue had decreased expression of Notchl. In training set, patients with high Notchl expression had better prognosis than those with low expression (49.5±2.4 vs 21.0±5.5 months, P<.001 for time to recurrence; 1-,3-year,100%,87%vs 86%, 43%, P<.001 for survival). Multivariate analysis indicated that Notchl expression level was an independent prognostic factor for both recurrence and survival (hazard ratio,95% confidence interval,4.310,2.108-8.813; 6.093,2.494-14.881, respectively). The results were fully confirmed in the analyses of validation set. Expression level of Notch1 was also an independent prognostic factor,for BCLC 0/A stage patients from two cohorts.Conclusion:Although expression of Notchl correlated positively with that of AAH, the mechanism of AAH was not through Notchl pathway. The expression level of Notchl in tumor tissue was closely associated with time to recurrence and survival of patients with early-stage hepatocellular carcinoma undergoing hepatectomy.In conclusion, overexpression of AAH was found in HCC tissues and cell lines, and was associated with proliferation and metastasis of HCC. Overexpression of AAH in hepatocellular carcinoma is associated with worse surgical outcomes. Axl was regulated by AAH, and the mechanism of AAH might be through Gas6/Axl pathway. Although Notchl could be regulated by AAH, the function of AAH was not through the Notch pathway. Notchl might play a tumor suppressor role in HCC and its expression level was linked to time to recurrence and survival of patients with early stage HCC.更多还原