5-脱氧杂氮胞苷对鼻咽癌Kank1基因去甲基化作用的研究

发布时间：2018-02-16 18:50

本文关键词： 鼻咽癌 Kank1基因 5-脱氧杂氮胞苷去甲基化　出处：《中南大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：本研究采用5-脱氧杂氮胞苷(5-Aza-CdR)对鼻咽癌不同细胞株进行处理,检测药物对细胞Kank1基因mRNA和蛋白表达的影响；检测Kank1基因启动子区甲基化变化情况；并分析肿瘤细胞生物学行为变化,为进一步探讨鼻咽癌的发生机制并寻找鼻咽癌治疗的新方法,同时对去甲基化药物5-脱氧杂氮胞苷在鼻咽癌中的应用做初步探索,为5-脱氧杂氮胞苷在临床中的应用提供新思路。方法：选用鼻咽癌细胞株5-8F,6-10B, CNE1, CNE2和正常鼻咽上皮细胞株NP69,再次验证Kank1基因在鼻咽癌细胞株中的低表达,将相对最低表达的6-10B, CNE1细胞株设为不加任何处理的对照组和经不同浓度5-Aza-CdR处理的实验组。利用实时荧光定量PCR及Western Blotting检测用药前后Kank1基因的表达变化；采用BSP克隆测序法检测Kank1基因启动子区CpG岛甲基化变化情况；同时检测用药前后细胞生物学行为变化,其中包括：相差显微镜观察各实验组细胞形态学改变情况；采用MTT比色法检测不同浓度5-Aza-CdR对细胞增殖的影响；利用流式细胞术检测不同浓度5-Aza-CdR处理72h后的细胞凋亡率。结果： 1.与正常鼻咽上皮细胞NP69相比,Kank1在鼻咽癌细胞株5-8F,6-10B, CNE1, CNE2中表达下调。 2.不同浓度5-Aza-CdR干预6-10B,CNE1细胞后其Kankl的表达得到不同程度上调,其表达上调程度与药物浓度成正比。 3. Western Blotting方法检测经10μM浓度5-Aza-CdR处理6-10B, CNE1细胞72h较未处理对照组其Kank1蛋白表达增强。 4.BSP克隆测序结果表明,经药物5-Aza-CdR处理的6-10B, CNE1细胞中Kank1基因启动子区域得到逆转,其平均甲基化百分数分别为11.11%、13.33%,明显低于未经药物处理组的甲基化化水平(分别为64.44%、64.44%,P0.01)。 5.MTT检测发现5-Aza-CdR可以明显抑制6-10B, CNE1细胞的生长与增殖。 6.流式细胞术检测结果表明细胞经药物处理后其凋亡率明显高于对照组,5-Aza-CdR能诱导细胞凋亡。结论： 5-Aza-CdR能够有效逆转鼻咽癌细胞株6-10B, CNE1中Kank1基因的异常甲基化,恢复Kank1mRNA和蛋白的表达,从而诱导肿瘤细胞凋亡,抑制鼻咽癌细胞生长。
[Abstract]:Objective: to investigate the effects of 5-deoxyazacytidine 5-Aza-CdR on the expression of Kank1 gene mRNA and protein in nasopharyngeal carcinoma (NPC) cells, and to detect the methylation of the promoter region of Kank1 gene. In order to explore the mechanism of nasopharyngeal carcinoma (NPC) and seek a new method for the treatment of nasopharyngeal carcinoma (NPC), the application of demethylated drug 5-deoxycytidine in nasopharyngeal carcinoma (NPC) was studied. To provide a new idea for the clinical application of 5-deoxyazacytidine. Methods: the low expression of Kank1 gene in nasopharyngeal carcinoma (NPC) cell lines 5-8FN6-10B, CNE1, CNE2 and NP69was confirmed again. The relative lowest expression of 6-10B, CNE1 cell line was divided into control group without any treatment and experimental group treated with 5-Aza-CdR at different concentrations. The expression of Kank1 gene was detected by real-time fluorescence quantitative PCR and Western Blotting. BSP clone sequencing method was used to detect the CpG island methylation in the promoter region of Kank1 gene, and the changes of cell biological behavior before and after treatment were also detected, including: phase contrast microscope was used to observe the changes of cell morphology in each experimental group. The effects of different concentrations of 5-Aza-CdR on cell proliferation were detected by MTT colorimetry, and the apoptotic rate of 5-Aza-CdR treated with different concentration of 5-Aza-CdR for 72 hours was detected by flow cytometry. Results:. 1.Compared with the normal nasopharyngeal epithelial cell line NP69, the expression of kank1 was down-regulated in the nasopharyngeal carcinoma cell lines 5-8FN6-10B, CNE1 and CNE2. 2. The expression of Kankl was up-regulated in different concentrations of 5-Aza-CdR, which was proportional to the drug concentration. 3. The expression of Kank1 protein in the CNE1 cells treated with 5-Aza-CdR at 10 渭 M for 72 h was higher than that in the untreated control group by Western Blotting assay. 4. The results of cloning and sequencing showed that the promoter region of Kank1 gene was reversed in 6-10Bcells treated with 5-Aza-CdR, and the average methylation percentage of Kank1 gene was 11.11and 13.33, respectively, which was significantly lower than that of untreated group (P 0.01). 5. MTT assay showed that 5-Aza-CdR could significantly inhibit the growth and proliferation of 6-10 B, CNE1 cells. 6. The results of flow cytometry showed that the apoptosis rate of the cells treated with drugs was significantly higher than that of the control group (5-Aza-CdR). Conclusion:. 5-Aza-CdR can effectively reverse the aberrant methylation of Kank1 gene and restore the expression of Kank1mRNA and protein in nasopharyngeal carcinoma cell line 6-10B, thus inducing apoptosis and inhibiting the growth of nasopharyngeal carcinoma cells.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.63

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