【作者】 孔令敏；

【导师】 陈志南； 邢金良；

【作者基本信息】 第四军医大学 ， 生物学， 2010， 博士

【摘要】 原发性肝癌(hepatocellular carcinoma, HCC)是世界常见恶性肿瘤,其每年发病600,000例,其中55%是在中国。因其恶性度高、病情进展快,治疗难度大、疗效差,五年生存率仅为8.9%。近二十年肝癌的治疗没有明显进展,所以急需一种新的预后评价指标,探索更为有效的治疗方法。CD147分子是一个高度糖基化的跨膜蛋白,它属于免疫球蛋白超家族,包含两个免疫球蛋白结构域(C2和I结构域)。CD147可以通过诱导MMPs分泌降解细胞外基质促进肿瘤侵袭转移,还可以通过VEGF、uPA、抑制失巢凋亡等途径促进肿瘤增殖转移。因此CD147是一个重要的癌生物标志物,对肝癌等恶性肿瘤诊断、预防及治疗都有着极其重要的意义。CD147的高表达常常和肿瘤的预后不良有关。虽然CD147在肿瘤的进展和预后中发挥重要的作用,但是其表达上调的分子机制还不清楚。转录水平的调控是真核生物基因表达调控中最重要的环节,而在转录起始阶段只有转录因子的特异结合才能有效起始转录。但是到目前为止能与CD147分子启动子区特异结合并能有效启动其转录的转录因子仍不清楚。CpG岛局部甲基化的异常变化往往早于细胞的恶性增生,全基因组的低甲基化伴随癌症的发生而出现,且随着肿瘤恶性程度的增加而更加明显,因此对DNA甲基化的检测可用于肿瘤诊断和预后评估,是癌症研究的热点之一。目前普遍认为的DNA甲基化参与基因表达调控的机制就是,甲基化直接的阻碍转录因子结合到含有CpG位点的结合区域。Sp1是序列特异的转录因子,通过结合到基因启动子区的GC富含区域来调节基因的表达。以前的研究表明Sp1结合位点的甲基化状态可影响Sp1的结合进而调控基因的转录。因此研究CD147启动子区CpG岛甲基化状态及其与肝癌的相关性,探讨甲基化对CD147的转录因子调控的影响,及甲基化程度与肝癌病人预后不良的相关性,对于理解肝癌的发生发展、早期诊断和靶向治疗有着重要的应用价值。本课题的研究目标有三点:首先确定CD147启动子核心区域及关键转录因子,分析转录因子对CD147的转录调控作用;其次分析CD147启动子区CpG岛是否存在甲基化现象,是否参与CD147的转录调控,了解各个甲基化位点在正常及肿瘤细胞系中的甲基化状态,及甲基化对转录因子结合是否存在影响;最后通过检测肝癌样本中甲基化状态来分析甲基化与肝癌患者预后的相关性,评价应用甲基化检测进行肝癌辅助诊断的可行性。第一部分:检测CD147分子在肝癌样本和肝癌细胞系的表达。我们运用免疫组化的方法检测了54例肝癌及其癌旁正常肝标本中CD147的表达和分布。结果显示CD147主要在表达在肝癌细胞中,而肝间质成纤维细胞不表达;在肝癌细胞内,染色主要定位于细胞膜和细胞浆上。CD147在肝癌组织中表达(42/54=77.78%)明显高于正常肝组织(4/54=7.4%,P<0.05)。然后我们运用实时定量PCR和蛋白免疫印迹的方法检测了正常肝组织,正常肝细胞和肝癌细胞中CD147的mRNA和蛋白表达水平。我们发现CD147分子的mRNA和蛋白水平在肝癌细胞中的表达均高于正常肝细胞,而且这种升高是发生在转录水平的。第二部分:确定CD147分子启动子转录核心区域。首先我们构建了含有CD147基因的启动子1.8kb长序列的荧光素酶报告载体,通过截短表达报告载体和瞬时转染检测萤火虫荧光素酶强度的方法确定CD147基因启动子核心区域。以CD147基因的转录起始位点为+1,确定了启动子的核心区域为-108到-42。同时利用转录因子预测软件预测,发现这个区域内含有一个转录因子Sp1的结合位点。第三部分:明确转录因子Sp1对CD147的转录调控作用。首先利用EMSA和ChIP的方法证明了转录因子Sp1在体外和体内均可结合到CD147基因的启动子区。同时我们也发现在CD147高表达的细胞HepG2细胞中Sp1结合到CD147启动子的量明显高于CD147低表达的细胞QZG。为进一步验证Sp1对CD147的转录激活作用,我们构建了Sp1结合位点突变的CD147基因启动子区的荧光素酶报告基因载体,发现CD147转录活性显著下降。过表达或者下调Sp1的表达可以改变CD147的转录活性。RNAi也证明了Sp1调节CD147的表达。因此我们认为转录因子Sp1可以结合到CD147启动子区并对其转录起着上调作用。第四部分:DNA甲基化修饰参与CD147基因表达调控。我们首先运用CpG岛搜索软件分析CD147启动子区域。我们发现在-340至+37(+1,CD147基因转录起始位点)是一个典型的CpG岛,同时这个区域亦包含了CD147的启动子核心区域。然后我们运用亚硫酸盐克隆测序的方法检测了正常肝组织,正常肝细胞系,肝癌细胞系中CD147启动子的甲基化情况。结果显示正常肝和正常肝细胞系CD147启动子区呈现较密集的甲基化状况,而肝癌细胞系中CD147启动子的甲基化程度就相对较低。进一步分析发现在Sp1的结合位点上在肝癌细胞系中甲基化程度明显低于正常对照。用去甲基化试剂5-Aza-dC处理细胞后,CD147表达水平随着5-Aza-dC作用浓度的不断升高而升高。而在此过程中Sp1的表达量没有发生明显的升高。同时ChIP检测发现5-Aza-dC处理提高了Sp1的结合量。反之过甲基化CD147启动子区会显著的下降CD147的转录活性。以上的结果提示,CD147启动子的低甲基化上调CD147基因在肝癌细胞的表达,这主要是由于去甲基化开放了转录因子的结合位点使CD147启动子结合更多的Sp1,进而上调CD147的表达。第五部分:CD147基因低甲基化与肝癌病人预后不良相关。我们运用亚硫酸盐克隆测序和甲基化特异PCR的方法分析了54例肝癌标本和其癌旁正常肝组织中CD147基因的甲基化情况。在肝癌标本中,CD147启动子甲基化程度与其蛋白表达呈负相关(r = -0.615),甲基化程度越高CD147表达越低。预后分析发现相对于发生CD147启动子甲基化的肝癌病人未发生CD147启动子甲基化的肝癌病人具有较高的复发率(88.1% vs. 58.3%;P<0.05)和死亡率(83.3% vs. 50.0%;P <0.05),提示肝癌病人CD147低甲基化与预后不良具有相关性,检测肝癌病人CD147甲基化情况可以作为肝癌病人预后评价的指标。根据上述结果,我们确定了CD147的转录核心区域及转录因子Sp1对其调控作用。CD147启动子区CpG岛存在甲基化调控的现象,甲基化调节参与CD147的转录调控,启动子低甲基化通过增加转录因子Sp1的结合上调CD147在肝癌细胞中的表达,这些结果有助于我们进一步理解肝癌发生发展的分子机制。这种低甲基化状况与肝癌病人预后不良存在相关性,提示检测CD147基因启动子甲基化状态,可用于肝癌的辅助诊断、监测肝癌患者的预后及治疗反应。干预CD147启动子甲基化,可作为肿瘤治疗的一种新策略。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with an annual incidence rate of about 600,000 cases, among which 55% are in China. HCC is characterized by vascular invasion, rapid progression and poor prognosis. The 5-year overall survival rate of individuals with HCC is only 8.9%, and this has barely improved over the past two decades. New prognostic markers are needed to help identify patients who are likely to have a poor prognosis and benefit from more aggressive treatment approaches.CD147 is a transmembrane protein with highly glycosylated modifications. It contains two extracellular immunoglobulin domains (C2 and I set), exhibiting the characteristics of the immunoglobulin superfamily members. CD147 expression significantly contributes to tumor progression by inducing the expression of MMPs (mainly MMP-2 and MMP-9) and vascular endothelial growth factor, activating the urokinase -type plasminogen activator system, conferring the resistance of tumor cells to anoikis and thus promoting tumor invasion and metastasis. Additionally, CD147 overexpression in tumor cells was significantly associated with poor prognosis. Nonetheless, although CD147 plays a critical role in tumor progression and prognosis, the mechanisms underlying the up-regulation of this molecule in cancer cells remain largely unknown.The gene expression has been shown to be dynamically regulated by many factors at both the transcriptional and posttranscriptional level. Among these mechanisms, the transcription factors seem to play the most crucial roles in gene regulation. Up to date, the cis-acting elements and transcription factors involved in CD147 expression was not been descripted. Aberrant methylation of CpG islands within promoter region usually happened earlier than cell hyperplasia; the global hypomethylation occurs with the cancer genesis and becomes more obviously with cancer development. So changes in DNA methylation patterns can be detected for diagnosis and prognosis.The most well-defined mechanism involved in the regulation of methylation-mediated gene expression is the direct binding blocking of transcription factors to their CpG-containing binding sites. Sp1 is a well-characterized sequence-specific transcriptional factor that regulates a large number of house-keeping and tissue-specific genes by binding to GC-rich DNA sequences in the promoter region of many human genes. Numerous previous studies have shown that methylation status of the Sp1 consensus binding site influences the Sp1 binding and thus regulates the gene transcription. The research in the association of methylation in HCC and its role in transcription regulation has significance in theory and application which helps understanding HCC genesis, development, early diagnosis and cancer biotherapy. The first purpose of our study was to identify the minimal promoter region of CD147 and the critical transcriptional factors. The second purpose was to analyze CpG islands within CD147 promoter, identify its potential methylated CG dinucleotide and methylation states in every site in normal liver and HCC cell lines, and investigate the transcription factor binding status. The third purpose was to investigate the association of methylation and HCC through detecting methylation states in generous HCC specimen, evaluate the feasibility of detecting methylation states as a new HCC diagnosis method.There were five parts in our study: (1) We detected the expression of cancer-associated molecule CD147 in HCC tissues and cell lines by immunohistochemical staining, real time RT-PCR and western blot; (2) We identified the minimal promoter region of CD147 by luciferase reporter assay and nested deletions of the CD147 promoter region; (3) With the assistance of the TRANSFAC database, the Sp1 binding sites were found in the critical promoter of CD147. Whether Sp1 could bind to the CD147 promoter was confirmed by point mutation experiment, in vivo and in vitro binding assays. These results were proven by blocking Sp1 using RNAi or mithramycin A treatment and upregulating Sp1 using transfection with eukaryotic expression vector; (4) CD147 promoter methylation statuses and related expression levels in normal and HCC cell lines and 54 paired HCC and adjacent non-tumor (ANT) tissues were respectively examined by bisulfite genomic sequencing, methylation-specific PCR, real-time RT-PCR, western blot and immunohistochemistry. Whether the binding of Sp1 to CD147 promoter could be influenced by methylation status was also investigated; (5) We characterized CD147 methylated sites in paraffin block specimen using methylation-specific PCR and BGS. For all HCC patients, follow-up analysis was carried out and the correlations of promoter methylation statuses with CD147 expression level, the clinicopathologic features and prognosis of patients were statistically analyzed.In this study, we explored the regulation of CD147 in HCC. The expression of CD147 was significant higher in HCC tissues than adjacent normal liver tissues. Over 80% of the HCC tissues expressed differential high levels of CD147. Then, we cloned the 5 -flanking region of human CD147 gene and identified a critical promoter region at -108 to -42 which contained one binding site for Sp1 (+1 as relative to the translation start site), which was essential in upregulating CD147 promoter activity. EMSA and ChIP assays demonstrated that Sp1 could bind to the CD147 promoter. These results were proven by blocking Sp1 using RNAi or mithramycin A treatment and upregulating Sp1 using transfection with eukaryotic expression vector. Significantly higher expression of CD147 and significantly lower promoter methylation level were observed in HCC cell lines compared to normal cell lines. Demethylation with 5-Aza-2 -deoxycytidine led to increased CD147 expression through enhancing Sp1 binding affinity and in vitro methylation of CD147 promoter reduced its transcriptional activity. CD147 promoter methylation level in HCC tissues (22.22%) was lower than that in ANT tissues (46.30%; P<0.05). Within HCC tissues, a significant inverse correlation was observed between CD147 expression and methylation level (r = -0.615). Moreover, HCC patients with unmethylated CD147 promoter had a significantly higher recurrence rate (88.1% vs. 58.3%; P<0.05) and death rate (83.3% vs. 50.0%; P <0.05) than patients with methylated CD147 promoter.In conclusion, our results suggest that Sp1 is essential for regulating the CD147 gene expression. We demonstrated methylation indeed presented in CpG island of CD147 promoter and regulated transcription of CD147. Promoter hypomethylation upregulates CD147 expression primarily through increasing Sp1 binding and associates with poor prognosis in HCC patients. These conclusions could help us improve our understanding the molecule machanism of HCC. Detecting the methylation state of CD147 promoter could be used for diagnosis and prognosis. Furthermore, intervening the methylation state of CD147 may become a new strategy for cancer therapy.更多还原