【作者】 苏波；

【导师】 李桂源；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 [研究背景]鼻咽癌是一种多步骤进展的多基因遗传性恶性肿瘤。目前,已发现鼻咽癌中越来越多的遗传学和表观遗传学分子改变,并且癌基因过表达或活性过高以及抑癌基因的表达缺失或失活可发生在不同临床／病理进展阶段。我们实验室经过多年的研究,发现数个候选抑瘤／易感基因参与多个细胞信号通路,并与鼻咽癌细胞增殖、侵袭和转移相关基因的表达有关。重要的是,这些基因表达缺失或失活同样也发生在鼻咽癌发生发展的不同阶段。上述研究表明,许多参与信号通路的遗传分子(尤其是组织特异性)相继激活或失活导致转录因子的活性以及所调控的靶基因表达异常,从而促进了鼻咽癌发生和发展。因此,基因异常表达谱与异常的转录调控模式密切相关。转录因子是基因调控网络中的重要分子。它接受上游信号通路异常信号,引起基因的表达紊乱,与肿瘤的发生发展密切相关。了解转录因子活性在肿瘤进展中的动态变化规律有利于阐明基因调控和表达异常的分子机制。尽管已有文献报道了某些转录因子在鼻咽癌组织或细胞中存在异常表达或活性改变,但是系统阐述转录因子活性在鼻咽癌不同临床进展阶段动态变化规律的研究尚无人问津。从而,本研究采用转录因子芯片分别检测和分析鼻咽癌不同临床进展阶段转录因子活性动态变化规律以及鼻咽癌细胞系与正常鼻咽上皮细胞系活性差异转录因子。[鼻咽癌不同临床进展阶段动态变化的转录因子活性谱的构建]对鼻咽癌不同临床进展阶段的组织样本进行检测,分析转录因子活性在临床进展中的动态变化。组织样本分为两组：1.独立样本组(12例),每例样本单独提取核蛋白,分别进行芯片检测；2.混合样本组(13例),同一临床阶段的样本混合,提取核蛋白后进行芯片检测。抽提临床Ⅰ-Ⅳ期组织核蛋白后,用Combo Protein/DNA芯片(含有345个检测位点)检测转录因子活性。采用One-way ANOVA和studentt检验方法获得55个差异转录因子。通过聚类分析发现26个转录因子在独立样本组和混合样本组中的活性变化趋势基本一致。这些转录因子在鼻咽癌临床进展阶段中活性增高并且呈动态性的变化。[AP2和ATF家族分子在鼻咽癌不同临床进展阶段的表达分析]通过线性回归分析,发现在26个活性增高的转录因子中,16个转录因子与鼻咽癌临床进展呈正相关性。这些转录因子分别是GAS/ISRE, CdxA/NKX2, HOXD8, PPUR, NFκB, AP2, Fra-1/JUN, AP3, PTF1, GKLF, ATF/CREB, RFX, C/EBP, Snail, PRDI-BFc和Stat5b。许多文献提示,AP2和ATF家族分子与多种肿瘤的发生有关。因此,我们选择这两个转录因子进行验证和分析。首先用EMSA证实了转录因子AP2和ATF的活性变化规律,进而扩大样本进一步对这两个转录因子家族的主要分子AP2α、AP2β、AP2γ、ATF1和ATF2以及靶基因EGFR和MMP-2进行免疫组化分析。采用Spearman’s rank test和Fisher’s exact test分析转录因子表达与鼻咽癌临床进展阶段和靶基因表达的相关性。结果显示,AP2α、AP2β、AP2γ、ATF1和ATF2在肿瘤组织中的表达明显高于正常鼻咽上皮,且与临床进展相关。AP2a与靶基因EGFR、ATF1和ATF2与靶基因MMP-2之间的表达具有相关性。Western blot和RT-PCR进一步验证了以上结果。[鼻咽癌细胞系活性差异转录因子分析]采用博奥生物技术有限公司的转录因子芯片(含有270个检测位点、)检测正常鼻咽上皮细胞系(NP69)、非转移性(6-10B)和转移性(5-8F)鼻咽癌细胞系中的转录因子活性。通过比较信号值,获得差异转录因子。鼻咽癌细胞中有10个转录因子上调,8个转录因子下调。值得注意的是,在上调的10个转录因子中,AP2,ATF/CREB, C/EBP和RFX与鼻咽癌组织的分析结果一致。与正常鼻咽上皮比较,它们在鼻咽癌组织中表达上调且与临床阶段的进展相关。与NP69相比,AP2, ATF/CREB和Spl的活性在两株鼻咽癌细胞中明显升高,EMSA分析进一步证实了芯片结果。RT-PCR和Western blot分析结果表明,AP2α、AP2β、AP2γ、ATF1、ATF2、Sp1和Sp3 mRNA表达水平在鼻咽癌细胞明显上调,其中AP2a、AP2γ、ATF1、ATF2、Sp1和Sp3蛋白水平在5-8F细胞高于6-10B细胞。此外,我们发现Spl、Sp3以及Spl靶基因VEGF和MMP-9在鼻咽癌组织高表达。[Spl对5-8F细胞侵袭能力的影响]为了明确Spl对5-8F细胞侵袭能力的影响,使用Sp1 siRNA转染细胞以干预Spl的表达。Sp1 siRNA能显著下调5-8F细胞中Spl的蛋白水平以及VEGF表达和MMP-9分泌。同时,用光辉霉素(一种Spl特异性的抑制剂)处理5-8F细胞后,发现光辉霉素能够呈剂量依赖性地抑制Sp1、VEGF和MMP-9的表达,同时5-8F细胞迁移和侵袭能力降低。这表明Spl表达和活性增高而引起MMP-9和VEGF的过表达,可能是5-8F细胞侵袭和转移能力增强的重要原因之一。综上所述,鼻咽癌不同临床阶段进展过程中转录因子活性动态变化规律的研究为我们进一步探索鼻咽癌相关基因转录调控机制提供了有价值的理论和实验基础。尽管目前我们得到了一些与临床进展相关的转录因子及其活性变化模式,但这并不代表我们揭示了所有与鼻咽癌发生发展密切相关的转录因子。本实验通过高通量的Protein/DNA芯片分析的确揭示了部分差异转录因子的活性变化规律,而这些转录因子在鼻咽癌进展过程中的动态变化规律可能与异常的基因表达谱密切相关。随着系统生物学和整合组学研究的进展,通过将不同临床进展阶段动态变化的转录因子差异活性谱与基因差异表达谱进行整合,将有利于阐明这些临床进展相关的差异转录因子对基因转录的调控机制,为鼻咽癌的诊断、预后提供检测分子标记以及临床治疗靶点。更多还原

【Abstract】 [Background]Nasopharyngeal carcinoma is characterized as a multiple-step, polygene-involved malignancy. More and more genetic and epigenetic changes have been verified. Overexpression or overactivation of oncogenes, and loss of expression or inactivation of tumor suppressor genes occur in different clinical progression stages. Based on the studies of NPC for many years, we previously have revealed that several tumor suppressor candidate genes inactivated in NPC played important roles in signaling pathways regulating gene expresson associated with proliferation, invasion and metastasis of NPC cells. More importantly, we have demonstrated that these molecular events (loss of expression or function) seem happen in different stages of NPC. These evidences described above suggested that many molecules involved in signaling pathways, especially tissue-specific, that were overactivated or inactivated successively may cause abnormal TF activation and gene expression, promoting the development and progression of NPC. The different gene expression profiles were highly associated with abnormal gene regulaion mechanism in NPC.TFs, which execute abnormal signal instructions from upstream molecules, are crucial modulators in gene regulation network, and act as critical roles in carcinogenesis. Understanding more knowledge about the laws of dynamic changes of TFs in tumor progression may facilitate to elucidate the underlying molecular mechanisms of gene regulation and explain aberrant gene expression modes from context. To date, some TFs overexpressed or overactivated in NPC tissue and cell lines have been reported, but there still lacks systematic data revealing activity change patterns of TFs in the process of this tumor. In this study, we profiled TFs activated in tissues from different clinical stages of NPC and in non-metastatic and metastatic cell lines by Protein/DNA arrays, and revealed differentially activated TFs showing kinetic changes in concert with the progression of this neoplasm.[The activity profile analysis of TFs in clinical stages]In this study, we attempted to obtain TF activity profiles and observe dynamic changes of TFs activated during the progression of NPC. The experiemental samples were divided into two sets, pooled set of 13 cases of which belongs to same clinical staging were pooled for array detection, and independent set of 12 cases, each of which was individually applied for array detection. Nuclear proteins extracted from clinical biopsy samples of stage I-IV patients were subjected to activity detection of TFs by Combo Protein/DNA arrays, which contained 345 DNA promoter binding sequences of TFs. We screened and selected 55 TFs showing differential activities between clinical stages in the independent set by one-way ANOVA and student t test. Comparative analysis between the two experiment sets revealed 26 TFs with similar change patterns by using clustering analysis, of which activities showed increase and dynamic alteration in the clinical progresssion.[The expression analysis of AP2 and ATF family TFs in NPC progression]Of the 26 TFs, we found 16 were up-regulated in association with clinical stages by a genealized linear model-based regression analysis. These TFs were GAS/ISRE, CdxA/NKX2, HOXD8, PPUR, NFκB, AP2, Fra-1/JUN, AP3, PTF1, GKLF, ATF/CREB, RFX, C/EBP, Snail, PRDI-Bfc and Stat5b. More and more evidences demonstrated have demonstrated that overacticated AP2 and ATF familys of TFs were implicated in carcinogenesis of diverse tumors. Thus, we selected AP2 and ATF for further identification in the follwing experiments. EMS A assays confirmed the results form analysis of Protein/DNA arrays. In the immunohistochemical analysis using extended clinical samples, we detected the expression of the main members of AP2 and ATF familys. Correlations between clinical stages and expression of TFs, and correlations of the expression of TFs with their target genes were analyzed using Fisher’s exact test and Spearman’s rank test. The results showed that AP2α, AP2β, AP2γ, ATF1 and ATF2 expression in nucleus increased in tumor tissues, and was associated with NPC progression. Moreover, AP2a, ATF1 and ATF2 expression was correlated with EGFR and MMP-2 expression, respectively. These results were further confirmed by Western blot and RT-PCR.[Transcription factors activated differentially in NPC cell lines]Another kind of Protein/DNA array containing 270 binding sites of TFs were employed to screen TFs showing differential activities between normal human nasopharyngeal epithelial cell line (NP69) and non-metastatic cell line (6-1 OB) and metastatic cell line (5-8F). By comparing the ratio of each spot in the arrays,10 upregulated TFs and 8 down-regulated TFs were identified in the two NPC cell lines. Of the up-regulated TFs, It was notable that AP2, ATF/CREB, C/EBP and RFX also showed increased activities in NPC tissues and association with clinical progression. Among the up-regulated TFs, AP2, ATF/CREB and Sp1 families displayed significantly differential activities in NPC cells compared with NP69 cells. The increased binding activities of AP2, ATF/CREB and Sp1 family were confirmed by EMS A. So, we explored the expression patterns of the main members of the three families of TFs in NPC cell lines by RT-PCR and Western blot. The specific primers of these genes were designed by using primer 3.0 software, sequences of primers and complying with BLAST procedure of NCBI online. The mRNA levels of AP2 proteins, ATF1, ATF2, Sp1 and Sp3 were markedly up-regulated in NPC cells. Notably, AP2a, AP2y, ATF1, ATF2, Sp1 and Sp3 protein expression in 5-8F was significantly higher than in 6-10B cells. In addition, Sp1, Sp3 and Sp1 target genes, VEGF and MMP-9 were overexpressed in NPC tissues.[Effect of Sp1 on invasive potential in 5-8F cells]To further determine whether Sp1 was involved in invasion and metastasis of 5-8F cells via up-regulating VEGF and MMP-9 expression, Sp1 siRNA was transfected into 5-8F cells. Sp1 siRNA significantly decreased Spl protein levels, VEGF expression and MMP-9 secretion. Then, a specific Sp1 inhibitor, mithramycin was applied to examine whether this agent have similar effects on 5-8F cells. The results showed that mithramycin effectively inhibited Sp1 expression in a dose-dependent manner, concomitant with decreased VEGF and MMP-9 expression in 5-8F cells. Moreover, reduced migrant and invasive potentialities were also observed in 5-8F cells. These data therefore indicate that high expression and activity of Sp1 may, at least in part, be important in promoting the potentiality of invasion and metastasis through up-regulating expression of MMP-9 and VEGF in 5-8F cells.In summary, the studies on the laws of dynamic activity changes of TF during NPC clinical progression provides a valuable platform for further researches on the mechanism of gene regulation. Although the features of the selected TFs that are characteristic of tumor progression could not uncover the activity state of all TFs that may be activated in this malignancy, In deed, the high-throughput Protein/DNA array analysis revealed the laws of kinetic activity changes of TFs that may cause aberrant gene expression profiles during this tumor progression. With the increase of information about systems biology and integromics, the integration of dynamic differential activity profiles of TFs with gene differential expression profiles, may facilitate to elucidate the mechanism of gene regulation by these TFs that correlated with clinic staging, thus providing potential diagnostic and prognostic tools, as well as therapeutic targets for NPC.更多还原