【作者】 潘奇；

【导师】 汤钊猷；

【作者基本信息】 复旦大学 ， 外科学， 2006， 博士

【摘要】 原发性肝癌是一种病死率很高的恶性肿瘤，是世界第三位、中国第二位的肿瘤死亡原因。手术治疗仍是目前肝癌最有效的治疗方法，但即使是根治术后5年转移复发率仍高达60-70％，因此术后高转移复发率是影响肝癌病人长期生存的关键。肿瘤转移是多基因参与，受多种信号转导蛋白和转录因子的调控的一个复杂过程。转录因子在肿瘤侵袭转移调控中起重要作用，近年来研究发现一些转录因子在一些肿瘤中表达及活性均明显异常，并且参与调控肿瘤细胞增殖、侵袭、血管生成等多种生物学功能。但目前转录因子的研究多集中在基因、蛋白表达水平，缺乏系统性转录因子功能研究，转录因子活性芯片的出现使系统研究转录因子活性与肝癌转移的关系成为可能。本课题第一部分拟用转录因子活性芯片分析不同转移潜能人肝癌细胞系细胞核内转录因子活性的差异，筛选与肝癌转移相关的关键转录因子，为肝癌转移复发研究提供新的预测指标和干预靶点。干扰素α是目前临床应用的能有效抑制肝癌转移复发的为数不多的药物之一。我所前期研究结果表明，干扰素α可抑制裸鼠肝癌的生长和转移，其作用机制是可能是通过下调血管内皮生长因子(VEGF)的转录和表达、进而抑制肿瘤血管生成发挥作用，但具体机制仍不是很清楚。有研究表明转录因子Sp1在VEGF基因调控中占有重要地位，本课题第二部分拟用VEGF启动子报告基因分析Sp1在干扰素α抑制高转移潜能肝癌细胞VEGF转录的作用，进一步研究干扰素α抑制肝癌血管生成和转移的作用机制，以便进一步提高干扰素预防转移复发的临床疗效。第一部分肝癌转移相关转录因子分析及活性差异转录因子的功能研究我所前期已建成具有不同转移潜能的人肝癌细胞模型体系。具有相似的遗传背景，但转移潜能具有明显的差别，是较好的对照研究系统。本课题第一部分对不同转移潜能人肝癌细胞系细胞核内转录因子进行了活性差异分析，筛选与肝癌转移相关的转录因子，并对筛选出来的转录因子进行了功能分析。我们首先应用转录因子活性芯片技术，在功能水平对三种不同转移潜能人肝癌细胞系Hep3B(无明显转移潜能)、MHCC97L(低转移潜能)和MHCC97H(高转移潜能)细胞核内转录因子活性谱进行差异分析，筛选出7个转录因子活性差异与人肝癌细胞转移潜能变化一致，其中有5个转录因子活性与肝癌细胞转移潜能呈正相关，包括p53、缺氧诱导因子-1α(HIF-1α)、核因子κb(NF-κb)、信号传导及转录活化因子3(Stat3)和Sp1；2个转录因子活性与肝癌细胞转移潜能呈负相关，包括Rb和Smad3，其中转录因子Sp1的活性差异采用凝胶电泳迁移率变动分析(EMSA)证实其活性与肝癌细胞转移潜能密切相关；接下来我们应用蛋白免疫印迹(Westem blot)和实时定量PCR(Real-Time PCR)又在转录、翻译水平和翻译后修饰水平进行了进一步分析，结果显示Sp1蛋白表达量和mRNA均随着转移潜能升高而显著增高，而Sp1蛋白磷酸化水平差异更为明显，这说明Sp1在肝癌组织中的过度表达和磷酸化异常是Sp1活性异常的原因，且与肝癌转移密切相关，提示Sp1可作为肝癌病人转移潜能的预测指标；最后我们采用RNA干扰技术沉默Sp1的基因表达，从而抑制Sp1蛋白表达，并进而抑制Sp1的活性，体外细胞生长抑制实验(MTT)实验显示，转染了Sp1 siRNA后，高转移潜能的MHCC97H细胞的生长能力较空白对照组和转染非特异性siRNA组有所下降，但没有显著性差异。而体外侵袭实验(Matrigel侵袭实验)表明抑制了Sp1的表达和活性后可明显降低肝癌细胞体外侵袭能力。提示特异性抑制Sp1活性或表达可抑制肝癌的侵袭性，进而抑制肝癌的转移复发。第二部分干扰素α抗肝癌转移的转录调控机制研究干扰素α是目前临床应用的能有效抑制肝癌转移复发的为数不多的药物之一。我所前期工作已证实干扰素α可抑制VEGF的转录和表达、进而抑制VEGF介导的肿瘤血管生成和转移，但具体的转录调控机制仍不清楚。有研究表明转录因子Sp1在VEGF基因调控中占有重要地位，本课题第拟用VEGF启动子报告基因分析Sp1在干扰素α抑制高转移潜能肝癌细胞VEGF转录的作用，进一步研究干扰素α抑制肝癌血管生成和转移的作用机制。首先我们构建VEGF全长启动子及5’端缺失突变体报告基因分析干扰素α对高转移潜能肝癌细胞(MHCC97H)的VEGF转录活性的作用，发现VEGF启动子上-109／-61区域是干扰素α抑制VEGF转录的最主要的调控区域，用转录因子活性芯片检测结合于VEGF启动子上的转录因子的活性，筛查出9个转录因子受到干扰素α作用，其中只有Sp1在-109／-61区域内有结合位点，进一步用不同转录因子结合位点点突变报告基因分析干扰素α对VEGF转录的作用，确定在此区域上的四个Sp1结合位点是干扰素α最主要的作用位点。我们应用RNA干扰技术发现下调Sp1的表达和活性后使干扰素α几乎丧失抑制VEGF转录的作用。接下来我们用凝胶电泳迁移率变动分析(EMSA)确定转录因子Sp1可特异性结合于VEGF启动子上，且干扰素α可明显的下调Sp1与VEGF启动子的结合活性，并呈剂量依赖性。说明干扰素α抑制VEGF转录的作用是通过下调Sp1活性起作用的。应用Westernblot分析干扰素α对MHCC97H细胞核内Sp1蛋白含量和磷酸化的作用，显示干扰素α明显下调了MHCC97H肝癌细胞核内Sp1的含量和磷酸化程度，其中磷酸化水平下调更为明显，说明干扰素α是通过下调Sp1在细胞核内含量和磷酸化程度抑制其与VEGF启动子上-109／-61区域的四个Sp1结合位点的结合活性，从而达到抑制VEGF介导的肿瘤血管生成和转移的。因此，我们可以说Sp1在干扰素α抗肝癌血管生成和转移中起重要的调控作用，提示Sp1可作为干扰素α临床疗效的预测指标。结论1．转移潜能不同肝癌细胞的转录因子活性谱存在显著差异。2．活性差异转录因子Sp1的活性、蛋白表达及磷酸化水平与肝癌转移潜能密切相关。3．干扰素α抑制VEGF介导的肝癌血管生成并由此抑制转移复发，主要是通过下调转录因子Sp1的表达或磷酸化，从而抑制其与VEGF启动子结合的活性而完成的。Sp1在干扰素α抗肝癌血管生成和转移中起重要的调控作用。潜在临床应用价值1．不同转移潜能肝癌细胞的活性差异转录因子的发现，有助于进一步了解肝癌转移机理，从另一个视野寻找预测指标和干预靶点。2．转录因子Sp1的活性、蛋白表达及磷酸化水平与肝癌转潜能密切相关，是肝癌病人转移复发的潜在预测指标和干预靶点。3．发现干扰素α通过下调Sp1抑制VEGF的转录和表达，为研究干扰素α的应用指征、耐药机制和联合用药提供了线索。4．Sp1在干扰素抗肝癌血管生成和转移中起重要的调控作用，提示Sp1可作为干扰素临床疗效的预测指标。创新点1．首次对不同转移潜能肝癌细胞转录因子活性进行系统分析，筛选出于肝癌转移相关的活性差异转录因子。2．首次探讨了干扰素α抑制肝癌细胞内VEGF及血管生成的转录调控机制。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is one of the most prevalent cancers with high fatality, which is the third highest cancer killer in the world, and is ranked second in China. The overall survival of patients with HCC is still unsatisfactory. Surgery remains the best treatment for a curative outcome of HCC, however, metastasis and recurrence is the major obstacle for further improve survival after surgery. Metastasis is a multigene-involved, multistep, and changing process, and regulated by various signaling pathways and transcription factors. Recently, overactivity and high expression of some transcription factors were found in nuclei of some malignant tumor cells, and act as modulators of cell proliferation, invasion, neo-angiogenesis, and metastasis. There are growing evidences show that transcription factors play a critical role in the invasion and metastasis of tumors by regulating transcription and expression of many metastasis associated genes. However, most of previous studies on transcription factor were focused primarily on protein and mRNA expression, and high-throughput functional analysis of multiple transcription factors was rarely reported. A novel technology - Protein/DNA array allows to profile the DNA binding activity of multiple transcription factors in a single array experiment, which make high-throughput functional analysis of multiple transcription factors possible. The aim of this study was to examine the activities of transcription factors in human HCC cell lines with different metastatic potentials, so as to identify transcription factors associated with HCC metastasis, and explore new predictive biomarkers and therapeutic targets.Interferon alpha (IFN-α) is one of few effective agents clinically used to prevent tumor metastasis. Previous studies from our institution found that IFN-α treatment dose dependently inhibits tumor growth and metastasis in metastatic nude mice model mediated by inhibition of VEGF gene transcription and expression, and resulted in inhibition of angiogenesis. However, the underlying mechanism remains unclear. Theaim of this study was to analyze the transcriptional mechanism of IFN-α on VEGF expression, angiogenesis and metastasis. So Sp1 plays a crucial role in the antiangiogenesis effect of IFN α, and may be a predictive biobarker of response of HCC patients to IFNα.Part oneTranscription factor activity profile of human hepatocellular carcinoma cell lines with different metastatic potentials and functional analysis of differential transcription factorsIn our institution, the establishment of a human HCC metastasis model system with similar genetic background and different metastatic potentials has provided a good platform for this study. This part is aimed to examine the activities of transcription factors in human HCC cell lines with different metastatic potentials, so as to identify transcription factors associated with HCC metastasis and analyze the biological activity of different transcription factors. Using Protein/DNA array, transcription factor activity profile of Hep3B, MHCC97L and MHCC97H, three HCC cell lines with different metastatic potentials, were examined. Seven activity differential transcription factors were found, 5 showed increased activity including p53、 hypoxia inducible factor-1 alpha (HIF-1α)、 signal transducer and activator of transcription 3 (Stat3) and Sp1, and 2 showed decreased activity including Rb and Smad3. Transcription factor Sp1 is associated with HCC metastasis and being confirmed by electrophoretic mobility shift assays (EMSA). Using western blot and real-time PCR, We detected Sp1 expression in transcription, translation and post-translation modification (phosphorylation, et al) level, and found that all of mRNA, protein expression, and phosphorylation status of Sp1 contributed to overactivity of Sp1 and also positively correlated to metastatic potential of HCC cells. Using RNA interference technique, we could specifically reduce expression level and DNA binding activity of Sp1 in MHCC97H cells (with high metastatic potential), and alteration of biological behavior of MHCC97H was found. MTT test showed that after transfecting of Sp1 siRNA, significant change of viability of MHCC97H cells was not observed. However, cell invasion assay in vitro showed that the decreased expression andactivity of Sp1 can significant decrease the invasiveness of MHCC97H cells.Part twoThe mechanism of the effect of interferon alpha on VEGF transcription and angiogenesisAs had mentioned, interferon alpha (IFN-α) can decrease vescular endothelial growth factor(VEGF) gene transcription and expression, and inhibit angiogenesis and metastasis. To elucidate the underlying transcriptional mechanism, this part will firstly determine the critical regions on VEGF responsible for the transcriptional inhibitory by IFN-α. A series of 5’ deletion mutants of VEGF promoter reporter gene based on the 2274-bp VEGF promoter were transfected into MHCC97H cells, and the effect of IFN-α on the VEGF promoter activity was examined. The result showed that the -109/-61 region contains essential regulatory elements. Secondly, to determine which transcription factor binding sites play the crucial role in regulation of VEGF promoter activity. The results showed that mutation in four Sp1 sites eliminated the inhibitory effect of IFN-α on VEGF promoter activity, whereas such effect was not found in mutation of either AP-2 or Egr-1 sites. It is suggested that Sp1 binding sites in this region are responsible for IFN-α-mediated inhibition of VEGF promoter activity. Thirdly, to determine whether inhibition of VEGF promoter activity by IFN-α is caused by changes in the interaction between nuclear protein and the promoter, protein/DNA array was performed using oligonucleotides corresponding to VEGF promoter as probe. It is demonstrated that IFN-α reduced the binding activity of 9 transcription factors, but only Sp1 has binding site in the -109/-61 region. EMS was performed using Sp1 consensus sequence and oligonucleotides corresponding to VEGF promoter region of nt -109 to-61, and found that Sp1 can bind to the region, and IFN-α treatment significantly reduced the binding activity of Sp1. Fourthly, to determine the role of Sp1 in inhibition of VEGF promoter activity by IFN-α, using RNA interference technique to reduce expression level and DNA binding activity of Sp1 in MHCC97H, and found that the inhibitory effect of IFN-α on VEGF promoter activity was almost eliminated. We then investigated whether the attenuation of Sp1 binding caused by IFN-α was due to decreased Sp1 protein expression and/orposttranslation modification. Western blot was performed, and found that IFN-α reduced both Sp1 protein level and Sp1 phosphorylation and decreased its DNA binding and transactivation activity.Conclusions1. There are some activity differential transcription factors in hepatocellular carcinoma cells with different metastatic potential.2. The activity and protein expression of transcription factor Sp1 is associated with metastatic potential of hepatocellular carcinoma.3. The inhibition effect of IFNα on VEGF-mediated tumor angiogenesis and metastasis is associated with the alteration of activity and protein expression of Spl.The potential application of this work1. The transcription factors, identified to associate with HCC metastasis, could be helpful to expand our understanding on mechanism of HCC metastasis and identify new predictive biomarkers and therapeutic targets.2. The association of transcription factor Sp1 with metastatic potential of hepatocellular carcinoma indicates that Sp1 might be a potential predictive biomarkers and therapeutic targets of HCC metastasis.3. The demonstration that IFN-α inhibits VEGF transcription mediated by downregulation of Sp1 could be of impact in patient selection, elucidation of drug resistance and designing combination treatment for using IFN-α to prevent HCC metastasis.The novelty of this work1. Demonstrate the transcription factor activity profile of human hepatocellular carcinoma cell lines with different metastatic potentials, and the association of Sp1 with HCC metastasis.2. Explore the mechanism of inhibitory effect of interferon alpha on VEGF transcription in HCC cell line.更多还原