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  本文選題：鼻咽癌 + R-cadherin基因��； 參考：《廣西醫(yī)科大學(xué)》2010年博士論文

【摘要】：鈣黏蛋白家族(Cadherinfamily)是一類重要的細(xì)胞黏附因子,其表觀遺傳學(xué)改變與腫瘤的浸潤和轉(zhuǎn)移有密切關(guān)系。在本實(shí)驗(yàn)研究中,我們用RT-PCR的方法檢測了鼻咽癌細(xì)胞株及腫瘤組織,正常鼻咽細(xì)胞株及正常鼻咽組織中R-cadherin基因的mRNA轉(zhuǎn)錄表達(dá)水平,并用甲基化特異性PCR的方法檢測了上述樣本中R-cadherin的DNA甲基化狀態(tài),我們發(fā)現(xiàn)4個鼻咽癌細(xì)胞株(CNE1、CNE2、C666-1、HONE1)和2個鼻咽癌移植瘤(C15,C17)中R-cadherin基因完全表達(dá)沉默,而在鼻咽癌細(xì)胞株TW03和正常鼻咽上皮永生化細(xì)胞株NP69的轉(zhuǎn)錄水平接近。鼻咽癌組織中R-cadherin基因的轉(zhuǎn)錄表達(dá)水平低于正常鼻咽組織中的水平;用甲基化特異性PCR檢測發(fā)現(xiàn)5個鼻咽癌細(xì)胞株、2個鼻咽癌移植瘤和94.3% (50/53)的鼻咽癌組織中R-cadherin基因?yàn)榧谆?而NP69與10例正常鼻咽上皮組織中則全為非甲基化。我們進(jìn)一步用5-aza-dC處理R-cadherin基因失活的兩個鼻咽癌細(xì)胞株,發(fā)現(xiàn)5-aza-dC能完全恢復(fù)這些細(xì)胞中R-cadherin基因的轉(zhuǎn)錄表達(dá),說明啟動子區(qū)域CpG島的高甲基化是鼻咽癌中R-cadherin基因轉(zhuǎn)錄表達(dá)失活的直接原因。為進(jìn)一步研究R-cadherin在鼻咽癌中的作用,我們從正常人腦組織中克隆了 R-cadherin基因的CDS全長,亞克隆入p-3×FLAG載體中,構(gòu)建了 p-3×FLAG-R-cadherin真核表達(dá)載體,并轉(zhuǎn)染R-cadherin表達(dá)沉默的鼻咽癌細(xì)胞株HONE1,通過篩選獲得穩(wěn)定轉(zhuǎn)染R-cadherin的鼻咽癌細(xì)胞株。經(jīng)過生長抑制實(shí)驗(yàn)、克隆形成實(shí)驗(yàn)、wound healing及熒光染料示蹤功能實(shí)驗(yàn)發(fā)現(xiàn),R-cadherin能抑制鼻咽癌細(xì)胞的增殖,減少鼻咽癌細(xì)胞克隆形成,抑制鼻咽癌細(xì)胞的遷移運(yùn)動及增加細(xì)胞間的縫隙連接水平。我們的結(jié)果顯示:鼻咽癌中R-cadherin基因因啟動子區(qū)DNA高甲基化而轉(zhuǎn)錄表達(dá)下調(diào),鼻咽癌中R-cadherin基因的高甲基化是一個腫瘤特異性的頻發(fā)事件;R-cadherin可抑制鼻咽癌細(xì)胞的惡性生物學(xué)行為,減緩鼻咽癌細(xì)胞的生長和侵襲,是鼻咽癌的候選抑癌基因。
[Abstract]:Cadherin family (cadherin family) is an important cell adhesion factor. Its epigenetic changes are closely related to tumor invasion and metastasis. In this study, RT-PCR was used to detect the expression of R-cadherin mRNA in nasopharyngeal carcinoma cell lines and tumor tissues, normal nasopharyngeal cell lines and normal nasopharyngeal tissues. The methylation status of R-cadherin was detected by methylation specific PCR. We found that the expression of R-cadherin gene was completely silenced in four NPC cell lines CNE1, CNE2C666-1, HONE1) and two nasopharyngeal carcinoma transplanted tumors, C15C17). The transcription level in nasopharyngeal carcinoma cell line TW03 and normal nasopharyngeal epithelial immortalized cell line NP69 was similar. The expression of R-cadherin gene in nasopharyngeal carcinoma was lower than that in normal nasopharyngeal tissue, and methylation of R-cadherin gene was found in 5 nasopharyngeal carcinoma cell lines, 2 nasopharyngeal carcinoma xenografts and 94.3% of nasopharyngeal carcinoma tissues by methylation specific PCR. NP69 and 10 normal nasopharyngeal epithelium were all demethylated. We further treated two nasopharyngeal carcinoma cell lines with R-cadherin gene inactivation with 5-aza-dC, and found that 5-aza-dC could completely restore the transcription expression of R-cadherin gene in these cells. It is suggested that hypermethylation of CpG island in promoter region is the direct cause of inactivation of R-cadherin gene expression in nasopharyngeal carcinoma. In order to further study the role of R-cadherin in nasopharyngeal carcinoma, we cloned the full-length CDS of R-cadherin gene from normal human brain tissue, subcloned it into p-3 脳 FLAG vector, and constructed p-3 脳 FLAG-R-cadherin eukaryotic expression vector. The NPC cell line HONE1, which was transfected with R-cadherin expression silencing, was transfected into the NPC cell line HONE1 and stable transfected with R-cadherin was obtained by screening. The results of growth inhibition test, clone formation test, healing and fluorescent dye tracing function test showed that R-cadherin could inhibit the proliferation of nasopharyngeal carcinoma cells and reduce the formation of nasopharyngeal carcinoma cell clone. Inhibit the migration of nasopharyngeal carcinoma cells and increase the level of gap junction between cells. Our results showed that the transcription expression of R-cadherin gene was down-regulated by DNA hypermethylation in the promoter region of nasopharyngeal carcinoma. Hypermethylation of R-cadherin gene in nasopharyngeal carcinoma was a tumor-specific frequent event that inhibited the malignant biological behavior of nasopharyngeal carcinoma cells. Slowing the growth and invasion of nasopharyngeal carcinoma cells is a candidate tumor suppressor gene for nasopharyngeal carcinoma.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R739.63

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本文編號：2043974