【作者】 殷欣；

【导师】 张博恒；

【作者基本信息】 复旦大学 ， 肿瘤学， 2011， 硕士

【摘要】 原发性肝癌是我国最常见的恶性肿瘤之一,其死亡率居全球第三位,在我国居恶性肿瘤第2位。虽然近年来肝癌研究已取得很大进展,以外科为主的综合治疗取得显著疗效,但根治性切除术后极高的复发转移率是影响肝癌疗效进一步提高的主要障碍。因此,探索肝癌复发转移的机制,寻找有效的靶向治疗是进一步提高肝癌患者长期生存的关键。2006年,Takahashi和Yamanaka将OCT4、SOX2、C-MYC和KLF4四个转录因子导入小鼠皮肤成纤维细胞.成功获得类似于胚胎干细胞(embryonic stem cells,ES细胞)的多能干细胞,命名为“诱导多能干细胞”(induced pluripotent stem cells.iPS细胞)。2007年,威斯康辛大学Thomson研究小组也报道了成功诱导胎儿体细胞转化为具有干细胞基本特征的人类iPS细胞,所不同的是他们使用慢病毒作为载体,选择了0CT4、SOX2、NANOG、LIN28等4个基因进行转录。这一类由成熟组织细胞经重新编程后转化而成的干细胞,被证实与真正的胚胎干细胞具有一样的分化潜能。肿瘤干细胞理论认为,肿瘤干细胞和胚胎干细胞之间具有许多共同特征,它们都具有自我复制,无限增殖,多向分化的潜能,相似的信号传导通路既调节胚胎干细胞也调节肿瘤干细胞的自我更新。可以推测,如果肿瘤的复发和转移源于肿瘤干细胞的存在,那么在干细胞中维持其分化增殖潜能的相关基因及其信号转导通路一定在肿,瘤的复发及转移中起着重要的作用。因此,如果肝细胞癌中存在这样一群0CT4、SOX2、C-MYC、KLF4、NANOG, LIN28高表达的细胞,这些细胞可能是具有自我更新和无限增殖的潜能的“干细胞样细胞”,而这样一群干细胞的存在必将成为肿瘤手术,放化疗后复发及转移的根源。本研究通过Western blot, Quantitative RT-PCR(Realtime RT-PCR)及免疫组化方法研究诱导多能干细胞转录因子OCT4、SOX2、C-MYC、KLF4、NANOG,LIN28在肝癌细胞系及肝癌组织标本中的表达,明确其在肝细胞癌复发及转移中的作用,进一步探索肝癌干细胞标志物,为肝癌的复发转移治疗提供新的治疗靶点。第一部分诱导多能干细胞转录因子在肝癌细胞系的表达目的：本研究旨在探索在肝正常细胞株中及不同侵袭转移潜能人肝癌细胞株中诱导多能干细胞转录因子的差异表达。方法：提取正常肝细胞株7701,低侵袭转移潜能细胞株HepG2, SMMC7721,MHCC97L,高转移潜能细胞株MHCC97H,HCCLM3细胞蛋白及RNA,通过Western blot及Realtime RT-PCR实验检测六个多能干细胞转录因子在不同转移潜能细胞株中的差异表达,明确其与肝细胞癌转移潜能的关系。结果：Western blot及Realtime RT-PCR证实,转录因子0CT4,NANOG在肝癌细胞株中,其表达水平随着肝癌转移潜能的升高而升高。差异具统计学意义(P<0.001,P=0.007),而SOX2的表达也随着肝癌转移潜能升高而升高,但差异不具统计学意义(P=0.12)。本实验证明了六个转录因子中,OCT4,SOX2,NANOG三个转录因子的阳性表达可能与肝细胞癌的转移潜能升高相关。第二部分诱导多能干细胞转录因子在肝癌组织中的表达及与肝癌切除术后复发的关系目的：研究诱导多能干细胞转录因子的表达与肝癌术后复发的关系方法：提取57例06年至08年在肝癌研究所行肝癌根治性切除的患者肿瘤组织中的RNA,根据术后是否出现复发分为2组：未复发组及复发组,进行Realtime RT-PCR实验,检测六个转录因子在复发及未复发患者肿瘤组织中的差异表达。结果：OCT4及NANOG mRNA的表达在复发组患者中高于非复发组,但样本量较小,差异未达到统计学意义。而SOX2,KLF4,C-MYC及LIN28mRNA在复发组患者的表达低于未复发组。本部分实验发现0CT4及NANOG mRNA在复发组患者中的表达高于未复发组患者,虽差异未达到统计学意义,但结合第一部分研究结果,提示OCT4及NANOG转录因子的阳性表达可能与肝细胞癌的复发转移相关。第三部分转录因子0CT4,NANOG与肝细胞癌术后复发转移,预后的关系目的：验证转录因子0CT4,NANOG与肝癌术后复发、转移、预后的关系。方法：选取2003年1月-2004年3月在复旦大学附属中山医院肝癌研究所进行手术切除、经病理确诊的肝细胞癌患者标本323例,制作组织芯片。通过免疫组化的方法观察OCT4,NANOG在肝癌组织芯片中的表达,研究其与肝细胞癌患者术后复发、转移、预后的关系。结果：323例患者癌旁的组织芯片中OCT4,NANOG的表达明显低于癌组织的表达。癌组织当中,OCT4胞核阳性表达的患者为78.4%,核表达率为0.5%-24.6%,平均7.6%；NANOG胞核阳性表达的患者为69.4%,核表达率为1%-12.4%,平均为5.4%。OCT4及NANOG的阳性表达与肝癌患者的AFP、GGT、肿瘤大小、数目、包膜、分化、癌栓、TNM分期均无明显关系,而0CT4及NANOG的联合阳性表达与肿瘤大小(P=0.03),血管侵犯相关(P=0.006)。生存分析提示0CT4及NANOG联合阳性表达的患者总体生存率明显低于阴性表达的患者,差异有统计学意义,前者术后因肿瘤死亡的患者死亡的风险为后者的1.9倍。单因素分析结果显示：0CT4阳性(P=0.03)、NANOG阳性(P=0.005)是肝细胞癌术后预后不良的重要影响因素。多因素分析提示OCT4及NANOG的独立阳性表达尽管不是肝癌预后的独立影响因素,但0CT4,NANOG共同阳性表达是肝细胞癌预后不佳的独立影响因素(P<0.001)。OCT4阳性的患者术后1年,3年,5年复发率高于OCT4阴性表达的患者,差异有统计学意义(P=0.006)。NANOG的阳性表达的患者术后复发率亦高于阴性表达的患者,但差异不具统计学意义(P=0.09)。OCT4及NANOG双阳性表达的患者复发率明显高于阴性表达的患者,差异有显著统计学意义(P<0.001)。多因素分析提示OCT4阳性/OCT4&NANOG阳性表达是肝癌术后复发的危险因素(P=0.005,P=0.007)。结论OCT4, NANOG为维持胚胎干细胞无限增殖,自我更新潜能信号通路中的关键转录因子。两个转录因子在高侵袭转移肝癌细胞系中表达明显增高,且其共同阳性表达与肝癌的复发,转移及预后差密切相关,提示肝癌组织可能中存在联合表达OCT4及NANOG的“肿瘤干细胞”样细胞,此类细胞具有无限增殖的潜能,可能是肝癌术后复发,转移的根源。OCT4及NANOG可能成为肝癌干细胞标志物及肝癌靶向治疗的新靶点。创新点1.首次系统地研究了六个诱导多能干细胞转录因子在肝癌细胞系及肝癌组织中的表达,并发现维持胚胎干细胞无限增殖潜能的关键转录因子OCT4及NANOG与肝癌的复发转移及预后不佳相关。2.为肝癌复发转移的治疗提供了新的治疗靶点更多还原

【Abstract】 Primary liver cancer is the most common malignancy in China. It is also recognized as the third cause of cancer death worldwide, and the second in China. Despite enormous progress made in the research field and surgical resection or orthotopic liver transplantation has been considered as the efficient therapy for patients with HCC, metastasis and recurrence remain the major obstacles for further prolonged survival. Elucidating molecular mechanisms of HCC recurrence/metastasis and searching for effective target therapies are the most important pathway to improve survival.In 2006, Yamanaka et al. first reprogrammed mouse somatic fibroblasts into induced pluripotent stem (iPS) cells by forced expression of four transcription factors:OCT4、SOX2、KLF4 and C-MYC. In 2007, Thomson’s group used the lentiviral transduction to reprogram human somatic cells to iPS with defined factors:OCT4、SOX2、NANOG、LIN28. Like ES cells, iPS cells have the potential to generate all the cell types of the body, and have important characteristics including self-renewal, pluripotency. These studies suggested OCT4、SOX2、C-MYC、KLF4、NANOG and LIN28 were the key genes for self-renewal, pluripotency.Based on cancer stem cell theory, cancer stem cells share many common features with embryonic stem cells such as potency of self-renewal, pluripotency. There is growing evidence of cross-talk and correlation between stemness pathways, tumor progression, and metastasis; If cancer stem cells are the origin of tumor recurrence and metastasis, the related signal pathway may play an important role in tumor progression. Cancer cells with high expression of OCT4、SOX2、C-MYC、KIF4、NANOG, LIN28 in HCC may be stem-like cells and probably are the seeds of recurrence and metastasis after radical treatment.To elucidate the role of OCT4、SOX2、C-MYC、KLF4、NANOG、LIN28 in hepatocellular carcinoma recurrence and metastasis, allow a better understanding of HCC initiation and progression, as well as establish precise targets for the development of more effective therapies, expression of transcription factors OCT4、SOX2、C-MYC、KLF4、NANOG,1IN28 were detected in normal hepatic cells、five HCC cell lines with different metastatic potentials and clinical tumor samples by Western blot, Realtime RT-PCR and immunohistochemistry method.PartⅠExpression of iPS transcription factors in hepatocellular carcinoma cell linesObjective:We detected the expression of OCT4、SOX2、C-MYC、KLF4、NANOG、LIN28 in hepatocellular carcinoma cell lines with Western blot and q RT-PCR.Methods:Total protein and RNA were extracted from normal hepatic cell line 7701 and HCC cell lines with different metastatic potentials. Expression of iPS transcription factors were detected by Western blot and Realtime RT-PCR.Results:Results from Western blot and Realtime RT-PCR showed high expression of OCT4, NANOG were leveled with increasing metastatic potential of cell lines. The different expression reached statistic significance (P<0.001, P=0.007). Expression of SOX2 also higher in high metastatic potential cell lines than low metastatic potential cell lines, but did not reach statistic significance (P=0.12).This study identified three potential iPS transcription factors correlated to invasive ability and metastasis in hepatocellular carcioma.PartⅡExpression of iPS transcription factors in hepatocellular carcinoma samples and their relationship with postoperative recurrence. Objective:The purpose of this study is to investigate the relationship between iPS transcription factors with postoperative recurrence in patients with hepatocellular carcinoma.Methods:Total RNA were extracted from clinical tumor samples of 57 patients undergoing surgical resection from 2006-2008 in Zhongshan Hospital, Liver Cancer Institute, Fudan University.57 patients were divided into two groups according to postoperative recurrence. mRNA expression of six iPS transcription factors were measured in two groups.Results:mRNA expression of OCT4 and NANOG were higher in recurrent group than non-recurrent group, although the difference did not reach statistic significance. The result suggested high expression of OCT4 and NANOG may be risk factors for postoperative recurrence.PartⅢRole of transcription factors OCT4 and NANOG in recurrence、metastasis and prognosis of hepatocellular carcinoma.Objective:To evaluate the expression of OCT4 and NANOG in hepatocellular carcinoma and discuss its clinical implications with recurrence、metastasis and prognosis.Methods:323 cases of HCC patients underwent radical resections from January 2003 to March 2004 in Zhongshan Hospital, Liver Cancer Institute, Fudan University. The corresponding blocks were manufactured into two groups of tissue microarray:group HCC and their corresponding tumor adjacent tissues. The expression of OCT4, NANOG were studied by immunohistochcmistry method. Difference between expression of OCT4, NANOG in HCC and adjacent liver tissues, and the relationship of the two iPS transcription factors with clinical pathological features and prognosis of HCC were analyzed. Results:Expression of OCT4 and NANOG were significantly higher in tumor tissues than that in tumor adjacent tissues.In tumor tissues,78.4% patients were found positively expressed of OCT4 in tumor cell nuclear and 69.4% patients were found positively expressed of NANOG. Expressive rate in nuclear were 0.5%-24.6% (mean:7.6%)for OCT4 and 1%-12.4%(mean:5.4%) for NANOG.There was no relationship between OCT4 or NANOG with clinical pathological features. Double positive expression of OCT4 and NANOG were found correlated with tumor size (P=0.03) and vascular invasion (P=0.006).Survival analysis revealed that co-expression of OCT4 and NANOG correlated to unfavorable overall survival after operation. As for patients with co-expression of OCT4 and NANOG, risk for postoperative death were 1.9 higher than that of negative patients. Although OCT4 and NANOG were identified important prognostic factors for survival in univariate analysis, they were not independent factors for prognosis in multivariate analysis. Co-expression of OCT4 and NANOG was an independent risk factor for unfavorable prognosis (P<0.001).Compared with OCT4 negative group, the 1,3,5 year recurrent rates were significantly higher in OCT4 positive group(P=0.006). The recurrent rates of NANOG positive group was also higher than NANOG negative group, but no difference were found with statistic significance (P=0.09). COX proportional hazard model identified expression of OCT4 (P=0.005) or co-expression of OCT4 and NANOG (P=0.007) were independent risk factor of postoperative recurrence.ConclusionsSelf-renewal and pluripotency are the central features in the definition of embryonic stem cells (ESC), in which OCT4 and NANOG play a key role in the maintenance of these processes. The preset study has demonstrated the expression of OCT4 and NANOG were correlated with recurrence, metastasis and poor prognosis in HCC. We speculate the two transcription factor can induce cancer stem cell-like properties and tumor cells with double positivity of OCT4 and NANOG may be stem-like cells, the seed of the tumorigenesis, recurrence, metastasis in HCC. We propose that OCT4 and NANOG would be potential markers of prognosis and novel targets of therapy for HCC.Novelty1. We first demonstrate iPS transcription factors OCT4 and NANOG, which are the key factors maintaining potency of self-renewal, pluripotency in ESC, attributed to recurrence/metastatic potentials in hepatocellular carcinoma.2.OCT4 and NANOG can be new targets for therapy inhibiting recurrence and metastasis after radical treatment in HCC.更多还原