食管鳞癌差异基因表达谱分析及HPGD在食管鳞癌中的初步研究

发布时间：2019-05-23 09:04

【摘要】：研究背景：我国是食管癌高发地区，其中超过90%的食管癌患者的病理类型为鳞状细胞癌。食管鳞癌的病因学及发病学机制迄今尚不明确。研究表明，食管鳞癌的发生和发展是一个多基因参与、多阶段诱变的过程，很多异常表达的基因在食管鳞状细胞的恶性转化过程中起了关键作用。从分子生物学水平研究食管鳞状细胞癌的发生发展，对食管鳞状细胞癌的诊断、预防和治疗具有重要的意义。基因芯片联合生物信息学技术，从整体上动态地、定量地观察肿瘤过程中关键基因的改变，能全面了解肿瘤发病机制，为肿瘤的预防及诊治提供新的思路。羟基前列腺素脱氢酶（hydroxyprostaglandin dehydrogenase，HPGD）是前列腺素降解的关键酶，同时对环氧合酶（cyclooxygenase, COX-2）具有生理性拮抗作用。近来研究显示，HPGD的表达缺失或减少可能与一些恶性肿瘤，如前列腺癌、结肠癌、胃癌、肺癌、乳腺癌、甲状腺癌、肝癌等的发生发展密切相关。研究目的: 1、通过基因芯片和深入的生物信息学分析技术，全面分析食管鳞癌事件进程中基因及相关通路等改变，以期找到该过程中起关键作用的基因及通路。 2、初步明确候选关键基因-HPGD对食管鳞癌增殖和侵袭等生物学作用，初步探讨其在致食管鳞癌事件中的可能机制。材料与方法: 1、收集外科手术切除的8对食管鳞癌和配对正常食管粘膜新鲜组织标本，应用基因芯片（Affymetric GeneChip Human Genome U133plus2.0Array）以及生物信息学分析寻找两组之间的差异表达基因（differentially expressed genes，DEGs），对筛查出的差异表达基因分别进行基因本体（Gene ontology，GO）功能富集度分析和Pathway功能分析，以及构建基因共表达网络。 2、对候选差异基因HPGD进一步功能验证和初步机制探讨。首先在线应用Oncomine、GEOdataset共享的芯片数据，荧光定量PCR及Western blot方法检测HPGD在食管癌组织及正常食管组织的表达情况。对60对福尔马林固定、石蜡包埋的食管鳞癌组织及配对癌旁正常组织标本作为研究对象。采用免疫组织化学的方法检测HPGD及COX-2蛋白在组织标本中的表达情况，并进行临床病理参数的相关性分析。以6株食管鳞癌细胞（TE1、TE2、TE8、KYSE150、KYSE410、EC9706）和一株正常食管上皮细胞株（Het-1A），采用荧光定量PCR方法和Western blot方法分别检测HPGD、COX-2的mRNA和蛋白的表达情况。筛选出相对高表达（TE1）和低表达（TE2）的食管癌细胞株。构建HPGD功能区全长表达载体以及靶向HPGD的shRNA载体，分别转染至相对高表达的细胞株和低表达的细胞株。分别利用荧光定量PCR和Western blot的方法检测HPGD及COX-2在转染前后mRNA及蛋白的表达变化。应用四甲基偶氮唑盐微量酶反应比色法(MTT)、BD bioCoatTMMatrigel侵袭小室等检测HPGD过表达或沉默后食管鳞癌细胞增殖和侵袭能力及相关基因的变化。应用甲基化特异性PCR（MSP）及亚硫酸盐盐测序方法（BSP）检测ESCC组织、癌旁组织及细胞株中HPGD启动子区域甲基化情况。应用去甲基化制剂5-氮杂脱氧胞（5-aza-dC）处理TE2细胞株后，应用RT-PCR方法检测mRNA表达情况。结果： 1、8对食管鳞癌组织及配对食管正常粘膜组织制备基因芯片后，绘制差异表达基因谱，共检出1208个差异表达基因，其中529个表达上调、679个表达下调。GO分析发现，上调差异基因显著性功能过程包括细胞外基质组成及分解、细胞周期、细胞粘附、细胞增殖等。下调基因主要参与小分子代谢、角化细胞分化、花生四烯酸代谢、钙离子依赖性细胞间粘附、上皮细胞增殖等过程。Pathway分析显示，上调差异基因参与的显著性信号转导通路是ECM受体相互作用通路、局部粘附通路、PI3K-Akt通路等，下调差异基因参与的显著性信号通路有代谢通路、花生四烯酸代谢通路、依赖细胞色素P450的药物代谢通路、细胞粘附分子（CAMs）通路。在构建的基因共表达网络中，我们筛选共表达地位变化大的差异基因，对其中之一HPGD拟进一步研究。 2、经验证HPGD在食管鳞癌组织中表达下调，与基因芯片相一致。免疫组化显示，HPGD在41例食管鳞癌组织呈阴性表达，COX-2在46例食管鳞癌组织中呈阳性表达，相关性分析显示两者在食管鳞癌表达呈负相关。HPGD蛋白的阴性表达、COX-2蛋白的阳性表达与食管鳞癌患者的淋巴结转移及是否累及外膜呈正相关(P＜0.05)，而与患者的性别、年龄、肿瘤细胞分化程度、肿瘤大小等无显著相关性（P＞0.05）。荧光定量PCR及Western blot发现HPGD在正常食管上皮细胞株Het-1A中表达，，在其他四个细胞株表达具有不同程度的表达下调。我们选择表达较低的ESCC细胞株TE2和表达较高的ESCC细胞株TE1。构建过表达及沉默HPGD的载体，分别转染至TE2和TE1细胞株。MTT显示，过表达HPGD后可抑制TE2细胞增殖，沉默HPGD表达后可促进TE1细胞增殖（P＜0.01）。Matrigel Transwell迁移和侵袭实验显示，过表达HPGD后可抑制TE2细胞迁移与侵袭，沉默HPGD表达后可促进TE1细胞迁移与侵袭（P＜0.01）。MSP、BSP检测Het-1A、TE8中HPGD启动子呈完全非甲基化，TE2细胞株呈完全甲基化，其他三株细胞株呈部分甲基化。TE2、EC9706细胞经去甲基化药物5-aza-dC处理后显示HPGD mRNA表达增加。结论： 1、经基因芯片联合生物信息学技术分析，筛选出HPGD是关键的下调差异表达基因之一，具有多种显著靶向性功能，参与肿瘤关键信号通路。 2、HPGD在食管鳞癌组织中存在表达缺失或下调，影响食管鳞癌细胞的增殖和侵袭能力。HPGD与COX-2在食管鳞癌中表达模式相反。HPGD食管鳞癌中发挥抑癌作用，启动子区DNA甲基化可能在食管鳞癌发生发展中起一定作用。
[Abstract]:Study Background: In the region of high incidence of esophageal cancer, more than 90% of the patients with esophageal cancer are of squamous cell type. The etiology and pathogenesis of squamous cell carcinoma of the esophagus are not yet unknown. The research shows that the occurrence and development of esophageal squamous cell carcinoma is a multi-gene and multi-stage mutation, and many abnormal expression genes play a key role in the malignant transformation of esophageal squamous cell. To study the development of esophageal squamous cell carcinoma from the level of molecular biology, it is of great importance to the diagnosis, prevention and treatment of esophageal squamous cell carcinoma. The gene chip is combined with the bioinformatics technology to dynamically and quantitatively observe the change of the key gene in the tumor from the whole, can comprehensively understand the pathogenesis of the tumor, and provide a new thinking for the prevention and the diagnosis and treatment of the tumor. The hydroxyprostaglandin dehydrogenase (HPGD) is a key enzyme for the degradation of prostaglandins, and has a physiological function on the cyclooxygenase (COX-2). Recent studies have shown that the deletion or reduction of the expression of HPGD may be associated with a number of malignant tumors, such as prostate cancer, colon cancer, gastric cancer, lung cancer, breast cancer, thyroid cancer, liver cancer, etc. Cutting-related. Objective: To study the changes of gene and related pathway in the process of esophageal squamous cell carcinoma by gene chip and in-depth bioinformatics analysis, in order to find a key role in this process. The biological effects of the candidate key gene-HPGD on the proliferation and invasion of esophageal squamous cell carcinoma were discussed. the incidence of cancer in the cancer event may Mechanisms, materials and methods:1,8 pairs of esophageal squamous cell carcinoma and paired normal esophageal mucosa fresh tissue specimens were collected, and the differential expression genes were found between the two groups using the gene chip (Affymetric GeneChip Human Genome U133plus2.0 Array) and the bioinformatics analysis. The gene ontology (GO) function enrichment analysis and the pathway function were carried out on the screened differentially expressed genes, respectively. And constructing a gene co-expression network. D. First, on-line application of Oncomine, GEOdataset shared chip data, fluorescence quantitative PCR and Western blot to detect the HPGD in the esophagus. The expression of cancerous tissue and normal esophageal tissue.60 cases of esophageal squamous cell carcinoma with formalin-fixed and paraffin-embedded tissues and its preparation The expression of HPGD and COX-2 protein in tissue specimens was detected by immunohistochemistry. The clinical and pathological parameters were analyzed. Six esophageal squamous cell lines (TE1, TE2, TE8, KYSE150, KYSE410, EC9706) and a normal esophageal epithelial cell line (Het-1A) were used. Expression of mRNA and protein of-2. Relative high expression (TE1) was selected. The invention discloses an esophageal cancer cell line with low expression (TE2), a full-length expression vector of the HPGD functional area and an shRNA vector targeting the HPGD are constructed, High-expression cell lines and low-expression cell lines, HPGD and COX-2 were detected by fluorescence quantitative PCR and Western blot respectively. The expression of mRNA and protein was detected before and after dyeing. The expression of HPGD and the expression of HPGD and the expression of HPGD were detected by the method of tetramethyl azo-salt microzyme reaction (MTT), BD bioCoatTMMatrigel invasion cell, etc. Proliferation and invasion ability and the change of related genes. The methylation specific PCR (MSP) and the sulfite salt sequencing method (BSP) were used to detect ESCC, adjacent tissues and cell lines. In that case of region methylation of the HPGD promoter, RT-PCR was used to treat the TE2 cell strain by demethylation preparation 5-aza-deoxy-cell (5-aza-dC). PCR Methods The expression of mRNA was detected. Results: After the gene chip was prepared by 1,8 pairs of esophageal squamous cell carcinoma and the normal mucosa of the paired esophagus, the differentially expressed genes were drawn, and 1208 differentially expressed genes were detected. Of these,529 were up-regulated and 679 down-regulated. GO analysis found that the significant sexual function of the up-regulated differential gene included extracellular matrix composition. and the down-regulation genes are mainly involved in small molecule metabolism, keratinocyte differentiation, arachidonic acid metabolism, calcium separation, Pathway analysis showed that the significant signal transduction pathway of the upregulation of the differential gene was the interaction pathway of the ECM, the local adhesion pathway, the PI3K-Akt pathway, and the significance of the down-regulation of the differential gene. The signal pathway has a metabolic pathway, a arachidonic acid metabolic pathway, a drug dependent on cytochrome P450, Metabolic pathway, cell adhesion molecule (CAMs) pathway. In the constructed gene co-expression network, we screened the difference in co-expression status heterogene, one of that HPGD is to be further study.2, validated HPGD The expression of COX-2 in esophageal squamous cell carcinoma (OSCC) was negative in 41 cases of esophageal squamous cell carcinoma, and the expression of COX-2 was positive in 46 cases of esophageal squamous cell carcinoma. The negative expression of the HPGD protein, the positive expression of the COX-2 protein and the lymph node metastasis of the patients with esophageal squamous cell carcinoma and the involvement of the adventitia were positively correlated (P <0.05). The expression of HPGD in the normal esophageal epithelial cell line Het-1A was found by fluorescence quantitative PCR and Western blot. The expression of the other four cell lines has a different degree of expression down-regulation. We choose to express a lower ESCC fine Cell strain TE2 and the high-expression ESCC cell line TE1. The expression and silencing of HP were constructed. The vector of GD was transfected into TE2 and TE1 cell lines respectively. MTT showed that after the expression of the HPGD, the proliferation of TE2 cells and the silence of the HPGD were inhibited. The proliferation of TE1 cells can be promoted after expression (P <0.01). Matrigel Transwell migration and invasion experiments show that after the expression of the HPGD, the migration and invasion of the TE2 cells can be inhibited, and the expression of the HPGD is silent. It can promote the migration and invasion of TE1 cells (P <0.01). The MSP and BSP test Het-1A, and the HPGD promoter in TE8 is completely non-methylated, and TE2 cells The strain was completely methylated and the other three cell lines were partially methylated. TE2 and EC9706 cells were demethylated 5-az. a-d The results showed that the expression of HPGD mRNA was increased after C treatment. One of the most significant targeted sexual functions involved in the tumor-critical signaling pathway. Lack of expression or down-regulation in the presence of expression, affecting the proliferation of esophageal squamous cell carcinoma cells and The expression pattern of the HPGD and the COX-2 in the esophageal squamous cell carcinoma is opposite to that of the expression pattern of the COX-2 in the squamous cell carcinoma of the esophagus.
【学位授予单位】：首都医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.1

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