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鼻咽癌细胞中miR-9表达下调的甲基化机制研究

发表时间:2017-03-15  浏览量:1100  下载量:252
全部作者: 罗花南,马思敬,易春曦,曹辉,郭海丽,侯瑾,闫静,任晓勇
作者单位: 西安交通大学第二附属医院耳鼻咽喉头颈外科病院;西安市中心医院耳鼻咽喉头颈外科;西安医学院第一附属医院耳鼻咽喉头颈外科
摘 要: 目的:研究miR-9在鼻咽癌细胞中表达下调是否与相应的编码基因启动子区DNA甲基化相关,明确miR-9表达失调的分子机制。方法:实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)检测分析正常鼻咽上皮细胞NP69和鼻咽癌细胞C666-1中加入去甲基化药物5-杂氮-2-脱氧胞苷(5-aza-2-deoxycytidine,5-AZA)(50 μmol/L,连续作用72 h)(NP69/C666-1 5-AZA组)和去乙酰化酶抑制剂曲古柳菌素A(trichostatin A,TSA)(10 nmol/L,继续作用24 h)(NP69/C666-1 5-AZA+TSA组)后细胞中miR-9的表达变化。同时,利用CpG Island Searcher软件对hsa-miR-9的编码基因hsa-miR-9-1、hsa-miR-9-2和hsa-miR-9-3启动子区的CpG岛进行分析,应用重亚硫酸盐测序(bisulfite genomic sequencing,BGS)检测5-AZA和TSA对hsa-miR-9-1、hsa-miR-9-2、hsa-miR-9-3启动子区CpG岛甲基化程度的影响。最后,应用CCK-8细胞增殖实验、流式细胞术、Transwell侵袭实验分析5-AZA和TSA对C666-1细胞增殖、凋亡和侵袭能力的影响。结果:与NP69对照组相比,NP69 5-AZA组和NP69 5-AZA+TSA组miR-9的表达水平和hsa-miR-9-1、hsa-miR-9-2、hsa-miR-9-3的甲基化率均无显著变化(P>0.05)。与C666-1对照组相比,C666-1 5-AZA组、C666-1 5-AZA+TSA组miR-9的表达水平分别上调11.31倍和22.63倍,差异有统计学意义(F=780.280,P=0.000)。BGS结果显示,C666-1对照组、C666-1 5-AZA组和C666-1 5-AZA+TSA组hsa-miR-9-1的甲基化率分别为78.8%(67/85)、32.9%(28/85)和14.1%(12/85),hsa-miR-9-2的甲基化率分别为72.2%(39/54)、38.9%(21/54)和14.8%(8/54),hsa-miR-9-3的甲基化率分别为76.0%(133/175)、32.0%(56/175)和20.6%(36/175),差异均有统计学意义(χ2=77.324,P=0.000;χ2=36.850,P=0.000;χ2=122.407,P=0.000)。5-AZA和TSA处理后,C666-1细胞的增殖和侵袭能力均显著下降(P<0.01)。结论:鼻咽癌细胞中miR-9表达下调与编码miR-9的基因miR-9-1、miR-9-2和miR-9-3启动子区CpG岛的甲基化有关。
关 键 词: 耳鼻咽喉科学;鼻咽癌;微小RNA;DNA甲基化;去甲基化药物
Title: Methylation mechanism of down regulated miR-9 expression in nasopharyngeal carcinoma cells
Author: LUO Huanan, MA Sijing, YI Chunxi, CAO Hui, GUO Haili, HOU Jin, YAN Jing, REN Xiaoyong
Organization: Department of Otolaryngology-Head and Neck Surgery, The Second Affiliated Hospital of Xi’an Jiaotong University; Department of Otolaryngology-Head & Neck Surgery, Xi’an Central Hospital; Department of Otolaryngology-Head & Neck Surgery, The First Affiliated Hospital of Xi’an Medical University
Abstract: Objective: To study whether the downregulation of miR-9 in nasopharyngeal carcinoma cells is related to DNA methylation in the promoter region of the coding gene and clarify the molecular mechanism of the disorder of miR-9 expression. Methods: In normal nasopharyngeal epithelial cell line NP69 and nasopharyngeal carcinoma cell line C666-1, quantitative real-time PCR (qRT-PCR) detection was used to analyze the effects of demethylation drug 5-aza-2-deoxycytidine (5-AZA, 50 μmol/L, 72 h) (NP69/C666-1 5-AZA group) and histone deacetylase inhibitor trichostatin A (TSA, 10 nmol/L, 24 h) (NP69/C666-1 5-AZA+TSA group) on the expression of miR-9. Meanwhile, the status of promoter CpG island methylation about hsa-miR-9-1, hsa-miR-9-2 and hsa-miR-9-3, which could encode hsa-miR-9, was analyzed by bisulfite genomic sequencing (BGS) after 5-AZA and TSA treatment. Finally, the effects of 5-AZA and TSA on the proliferation, apoptosis and invasion abilities of C666-1 cells were analyzed by CCK-8 cell proliferation assay, flow cytometry and Transwell invasion assay. Results: The expression levels of miR-9 and hsa-miR-9-1, hsa-miR-9-2 and hsa-miR-9-3 methylation rates in NP69 5-AZA group and NP69 5-AZA+TSA group were almost the same as those in the NP69 group (P>0.05). The expression levels of miR-9 were up 11.31 times and 22.63 times in C666-1 5-AZA group and C666-1 5-AZA+TSA group respectively, compared with those in the C666-1 group, and the differences were statistically significant (F=780.280, P=0.000). BGS detection showed that in C666-1, C666-1 5-AZA and C666-1 5-AZA+TSA groups, the methylation ratios of hsa-miR-9-1 were 78.8% (67/85), 32.9% (28/85) and 14.1% (12/85), those of hsa-miR-9-2 and hsa-miR-9-3 were 72.2% (39/54), 38.9% (21/54) and 14.8% (8/54), 76.0% (133/175), 32.0% (56/175) and 20.6% (36/175), respectively, and the differences were statistically significant (χ2=77.324, P=0.000; χ2=36.850, P=0.000; χ2=122.407, P=0.000). The proliferation and invasion abilities of C666-1 cells were significantly decreased after 5-AZA and TSA treatment (P<0.01). Conclusion: The downregulation of miR-9 expression in nasopharyngeal carcinoma cells is associated with CpG island methylation in the promoter regions of miR-9 encoding genes including miR-9-1, miR-9-2 and miR-9-3.
Key words: otorhinolaryngology; nasopharyngeal carcinoma; microRNA; DNA methylation; demethylation drugs
发表期数: 2017年3月第5期
引用格式: 罗花南,马思敬,易春曦,等. 鼻咽癌细胞中miR-9表达下调的甲基化机制研究[J]. 中国科技论文在线精品论文,2017,10(5):493-501.
 
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