本文選題：TFPI-2 + 鼻咽癌　； 參考：《廣西醫(yī)科大學(xué)》2011年碩士論文

【摘要】：鼻咽癌在世界大部分地區(qū)是一種罕見(jiàn)的疾病,但在中國(guó)南方地區(qū)是最常見(jiàn)并致病人死亡的頭頸部惡性腫瘤之一。過(guò)往研究顯示,不同于其他腫瘤中經(jīng)典癌基因發(fā)生缺失或突變,鼻咽癌更傾向于是一個(gè)表觀遺傳學(xué)疾病。鼻咽癌特異性腫瘤抑癌基因的啟動(dòng)子區(qū)CpG島異常甲基化導(dǎo)致其轉(zhuǎn)錄表達(dá)下調(diào)或沉默,喪失腫瘤抑制功能,成為鼻咽癌發(fā)病的重要機(jī)制。 我們?cè)谇捌谘芯恐?利用甲基轉(zhuǎn)移酶抑制劑聯(lián)合脫乙酰基酶抑制劑處理鼻咽癌兩個(gè)細(xì)胞株CNE2和HONE1,運(yùn)用高通量的cDNA表達(dá)芯片篩選因啟動(dòng)子區(qū)DNA甲基化而表達(dá)下調(diào)的候選抑癌基因。其中組織因子途徑抑制物2(tissue factor pathway inhibitor-2,TFPI-2)的轉(zhuǎn)錄表達(dá)水平上調(diào)明顯,在CNE2和HONE1兩個(gè)細(xì)胞株中分別上調(diào)了107和49倍。這些結(jié)果提示我們TFPI-2基因在鼻咽癌中可能是一個(gè)表觀遺傳失活的腫瘤抑制基因,本實(shí)驗(yàn)將對(duì)TFPI-2在鼻咽癌中轉(zhuǎn)錄表達(dá)失活發(fā)生機(jī)制、以及TFPI-2基因的抑癌功能進(jìn)行研究。 我們通過(guò)RT-PCR(reverse transcriptase PCR)技術(shù)檢測(cè)TFPI-2基因mRNA轉(zhuǎn)錄表達(dá)水平,結(jié)果顯示:在6個(gè)鼻咽癌細(xì)胞株CNE1,CNE2,TW03,C666-1,HNE1和HONE1中,TFPI-2 mRNA表達(dá)下調(diào)或沉默,在正常鼻咽上皮組織則高表達(dá)。運(yùn)用甲基化特異性PCR(MSP,Methylation Specific PCR),TFPI-2啟動(dòng)子區(qū)CpG島高甲基化可在66.7%(4/6)的鼻咽癌細(xì)胞株及88.6%(62/70)的鼻咽癌組織DNA中檢測(cè)到,而未見(jiàn)于正常鼻咽上皮組織(0/12)。進(jìn)一步運(yùn)用甲基轉(zhuǎn)移酶抑制劑5-aza-dC(5-aza-2-deoxycytidine)對(duì)3株鼻咽癌細(xì)胞株去甲基化處理后,發(fā)現(xiàn)TFPI-2在鼻咽癌細(xì)胞株的表達(dá)均發(fā)生上調(diào)或恢復(fù)表達(dá),說(shuō)明鼻咽癌中TFPI-2基因啟動(dòng)子區(qū)CpG島高甲基化是其表達(dá)失活的直接原因。 為明確TFPI-2在鼻咽癌中的作用,我們從TureClone人TFPI-2全長(zhǎng)cDNA (Origene,USA)中擴(kuò)增出長(zhǎng)為708bp的TFPI-2基因編碼區(qū)全長(zhǎng),構(gòu)建其真核表達(dá)載體,并將其轉(zhuǎn)染至不表達(dá)TFPI-2的鼻咽癌細(xì)胞系,篩選獲得了穩(wěn)定轉(zhuǎn)染TFPI-2的細(xì)胞株。通過(guò)生長(zhǎng)抑制實(shí)驗(yàn)、克隆形成實(shí)驗(yàn)、遷移運(yùn)動(dòng)試驗(yàn)及細(xì)胞凋亡分析,提示TFPI-2可有效抑制鼻咽癌細(xì)胞的生長(zhǎng)、降低細(xì)胞克隆形成效率,抑制細(xì)胞的遷移運(yùn)動(dòng)能力并誘導(dǎo)鼻咽癌細(xì)胞的凋亡。 我們的實(shí)驗(yàn)結(jié)果表明TFPI-2基因在鼻咽癌中因?yàn)閱?dòng)子區(qū)CpG島DNA高甲基化而轉(zhuǎn)錄失活;體外表達(dá)TFPI-2可逆轉(zhuǎn)鼻咽癌細(xì)胞的惡性生物學(xué)行為并誘導(dǎo)腫瘤細(xì)胞凋亡;因此,TFPI-2是一個(gè)鼻咽癌相關(guān)的潛在腫瘤抑制基因。
[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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R739.63

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相關(guān)期刊論文 前1條

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本文編號(hào)：1838214