TFPI-2基因在鼻咽癌中转录失活机制及其抑癌功能验证

发布时间：2018-05-03 10:54

本文选题：TFPI-2 + 鼻咽癌　；参考：《广西医科大学》2011年硕士论文

【摘要】：鼻咽癌在世界大部分地区是一种罕见的疾病,但在中国南方地区是最常见并致病人死亡的头颈部恶性肿瘤之一。过往研究显示,不同于其他肿瘤中经典癌基因发生缺失或突变,鼻咽癌更倾向于是一个表观遗传学疾病。鼻咽癌特异性肿瘤抑癌基因的启动子区CpG岛异常甲基化导致其转录表达下调或沉默,丧失肿瘤抑制功能,成为鼻咽癌发病的重要机制。我们在前期研究中,利用甲基转移酶抑制剂联合脱乙酰基酶抑制剂处理鼻咽癌两个细胞株CNE2和HONE1,运用高通量的cDNA表达芯片筛选因启动子区DNA甲基化而表达下调的候选抑癌基因。其中组织因子途径抑制物2(tissue factor pathway inhibitor-2,TFPI-2)的转录表达水平上调明显,在CNE2和HONE1两个细胞株中分别上调了107和49倍。这些结果提示我们TFPI-2基因在鼻咽癌中可能是一个表观遗传失活的肿瘤抑制基因,本实验将对TFPI-2在鼻咽癌中转录表达失活发生机制、以及TFPI-2基因的抑癌功能进行研究。我们通过RT-PCR(reverse transcriptase PCR)技术检测TFPI-2基因mRNA转录表达水平,结果显示:在6个鼻咽癌细胞株CNE1,CNE2,TW03,C666-1,HNE1和HONE1中,TFPI-2 mRNA表达下调或沉默,在正常鼻咽上皮组织则高表达。运用甲基化特异性PCR(MSP,Methylation Specific PCR),TFPI-2启动子区CpG岛高甲基化可在66.7%(4/6)的鼻咽癌细胞株及88.6%(62/70)的鼻咽癌组织DNA中检测到,而未见于正常鼻咽上皮组织(0/12)。进一步运用甲基转移酶抑制剂5-aza-dC(5-aza-2-deoxycytidine)对3株鼻咽癌细胞株去甲基化处理后,发现TFPI-2在鼻咽癌细胞株的表达均发生上调或恢复表达,说明鼻咽癌中TFPI-2基因启动子区CpG岛高甲基化是其表达失活的直接原因。为明确TFPI-2在鼻咽癌中的作用,我们从TureClone人TFPI-2全长cDNA (Origene,USA)中扩增出长为708bp的TFPI-2基因编码区全长,构建其真核表达载体,并将其转染至不表达TFPI-2的鼻咽癌细胞系,筛选获得了稳定转染TFPI-2的细胞株。通过生长抑制实验、克隆形成实验、迁移运动试验及细胞凋亡分析,提示TFPI-2可有效抑制鼻咽癌细胞的生长、降低细胞克隆形成效率,抑制细胞的迁移运动能力并诱导鼻咽癌细胞的凋亡。我们的实验结果表明TFPI-2基因在鼻咽癌中因为启动子区CpG岛DNA高甲基化而转录失活;体外表达TFPI-2可逆转鼻咽癌细胞的恶性生物学行为并诱导肿瘤细胞凋亡;因此,TFPI-2是一个鼻咽癌相关的潜在肿瘤抑制基因。
[Abstract]:Nasopharyngeal carcinoma is a rare disease in most parts of the world, but it is one of the most common and fatal head and neck malignancies in southern China. Previous studies have shown that nasopharyngeal carcinoma is more prone to epigenetic disease than classic oncogene deletion or mutation in other tumors. The abnormal methylation of CpG island in the promoter region of the tumor suppressor gene of nasopharyngeal carcinoma (NPC) leads to down-regulation or silencing of its transcription and loss of tumor suppressor function, which is an important mechanism in the pathogenesis of nasopharyngeal carcinoma (NPC). In our previous study, two nasopharyngeal carcinoma cell lines, CNE2 and HONE1, were treated with methyltransferase inhibitor and deacetylase inhibitor, and candidate tumor suppressor genes down-regulated by promoter region DNA methylation were screened by high-throughput cDNA expression microarray. The expression of tissue factor pathway inhibitor 2(tissue factor pathway inhibitor-2TFPI-2 was significantly up-regulated in CNE2 and HONE1 cells by 107-fold and 49-fold, respectively. These results suggest that our TFPI-2 gene may be an epigenetic inactivated tumor suppressor gene in nasopharyngeal carcinoma. In this study, we will study the mechanism of transcription inactivation of TFPI-2 in nasopharyngeal carcinoma and the inhibitory function of TFPI-2 gene. We detected the mRNA transcription level of TFPI-2 gene by RT-PCR(reverse transcriptase PCR technique. The results showed that the expression of TFPI-2 mRNA was down-regulated or silenced in 6 NPC cell lines CNE1, CNE2C666-1HNE1 and HONE1, but was overexpressed in normal nasopharyngeal epithelium. Hypermethylation of CpG island in the promoter region of TFPI-2 was detected in nasopharyngeal carcinoma cell lines (66.7 / 6) and nasopharyngeal carcinoma (88.6 / 62 / 70), but not in normal nasopharyngeal epithelial tissue (0 / 1212). Further treatment with methyltransferase inhibitor 5-aza-dCon 5-aza-2-deoxycytidine showed that the expression of TFPI-2 in nasopharyngeal carcinoma cells was up-regulated or restored after demethylation. It was suggested that hypermethylation of CpG island in the promoter region of TFPI-2 gene was the direct cause of inactivation of NPC expression. In order to clarify the role of TFPI-2 in nasopharyngeal carcinoma (NPC), we amplified the full length of TFPI-2 gene coding region from TureClone human TFPI-2 cDNA, constructed its eukaryotic expression vector, and transfected it into nasopharyngeal carcinoma cell line without TFPI-2 expression. The cell lines stably transfected with TFPI-2 were obtained. The results of growth inhibition test, clone formation test, migration exercise test and apoptosis analysis showed that TFPI-2 could effectively inhibit the growth of nasopharyngeal carcinoma cells and reduce the efficiency of cell clone formation. Inhibit cell migration and motor ability and induce apoptosis of nasopharyngeal carcinoma cells. Our results indicate that TFPI-2 gene is inactivated in nasopharyngeal carcinoma due to the hypermethylation of DNA in the promoter region of CpG island, and the expression of TFPI-2 in vitro can reverse the malignant biological behavior of nasopharyngeal carcinoma cells and induce apoptosis of tumor cells. Therefore, TFPI-2 is a potential tumor suppressor gene associated with nasopharyngeal carcinoma.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.63

【参考文献】