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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：1544 下载量：440
| 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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