鼻咽癌中R-cadherin基因的表观沉默机制及抑癌功能研究

发布时间：2018-06-20 10:40

本文选题：鼻咽癌 + R-cadherin基因　；参考：《广西医科大学》2010年博士论文

【摘要】：钙黏蛋白家族(Cadherinfamily)是一类重要的细胞黏附因子,其表观遗传学改变与肿瘤的浸润和转移有密切关系。在本实验研究中,我们用RT-PCR的方法检测了鼻咽癌细胞株及肿瘤组织,正常鼻咽细胞株及正常鼻咽组织中R-cadherin基因的mRNA转录表达水平,并用甲基化特异性PCR的方法检测了上述样本中R-cadherin的DNA甲基化状态,我们发现4个鼻咽癌细胞株(CNE1、CNE2、C666-1、HONE1)和2个鼻咽癌移植瘤(C15,C17)中R-cadherin基因完全表达沉默,而在鼻咽癌细胞株TW03和正常鼻咽上皮永生化细胞株NP69的转录水平接近。鼻咽癌组织中R-cadherin基因的转录表达水平低于正常鼻咽组织中的水平;用甲基化特异性PCR检测发现5个鼻咽癌细胞株、2个鼻咽癌移植瘤和94.3% (50/53)的鼻咽癌组织中R-cadherin基因为甲基化,而NP69与10例正常鼻咽上皮组织中则全为非甲基化。我们进一步用5-aza-dC处理R-cadherin基因失活的两个鼻咽癌细胞株,发现5-aza-dC能完全恢复这些细胞中R-cadherin基因的转录表达,说明启动子区域CpG岛的高甲基化是鼻咽癌中R-cadherin基因转录表达失活的直接原因。为进一步研究R-cadherin在鼻咽癌中的作用,我们从正常人脑组织中克隆了 R-cadherin基因的CDS全长,亚克隆入p-3×FLAG载体中,构建了 p-3×FLAG-R-cadherin真核表达载体,并转染R-cadherin表达沉默的鼻咽癌细胞株HONE1,通过筛选获得稳定转染R-cadherin的鼻咽癌细胞株。经过生长抑制实验、克隆形成实验、wound healing及荧光染料示踪功能实验发现,R-cadherin能抑制鼻咽癌细胞的增殖,减少鼻咽癌细胞克隆形成,抑制鼻咽癌细胞的迁移运动及增加细胞间的缝隙连接水平。我们的结果显示:鼻咽癌中R-cadherin基因因启动子区DNA高甲基化而转录表达下调,鼻咽癌中R-cadherin基因的高甲基化是一个肿瘤特异性的频发事件;R-cadherin可抑制鼻咽癌细胞的恶性生物学行为,减缓鼻咽癌细胞的生长和侵袭,是鼻咽癌的候选抑癌基因。
[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.
【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R739.63

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