启动子甲基化调节在低表达环氧合酶-2的食管鳞癌移植模型中的作用

doi:10.3969/j.issn.1006-7795.2014.06.022

首都医科大学学报 ›› 2014, Vol. 35 ›› Issue (6): 798-804.doi: 10.3969/j.issn.1006-7795.2014.06.022

启动子甲基化调节在低表达环氧合酶-2的食管鳞癌移植模型中的作用

王青釭, 孟莹, 朱圣韬, 施海韵, 张澍田

首都医科大学附属北京友谊医院消化内科首都医科大学消化病学系北京市消化疾病中心, 北京 100050

收稿日期:2014-03-28 发布日期:2014-12-15
通讯作者: 张澍田 E-mail:zhangst@ccmu.edu.cn
基金资助:
国家自然科学基金(81302160),国家重点基础研究发展计划(973计划)(2012CB526600),北京市自然科学基金(KZ201410025024)

Effect of promoter methylation on lower expressed cyclooxygenase-2 esophageal squamous cell carcinoma xenograft

Wang Qinggang, Meng Ying, Zhu Shengtao, Shi Haiyun, Zhang Shutian

Department of Gastroenterology, Beijing Friendship Hospital, Department of Digestive Disease, Capital Medical University, Beijing Digestive Disease Center, Beijing 100050, China

Received:2014-03-28 Published:2014-12-15
Supported by:
This study was supported by National Natural Science Foundation of China(81302160), National Key Basic Research Program of China(973 Program)(2012CB526600), Natural Science Foundation of Beijing(KZ201410025024).

摘要/Abstract

摘要：

目的环氧合酶-2(cyclooxygenase-2,COX-2) 参与食管鳞癌(esophageal squamous cell carcinoma,ESCC)的发生发展过程,但在部分ESCC细胞株中COX-2呈低表达。选择低表达COX-2基因的ESCC进行裸鼠移植。探讨COX-2基因甲基化与基因表达的关系,观察联合COX-2选择性抑制剂和去甲基化药物干预对移植瘤生长的影响。方法选择KYSE 150细胞进行裸鼠移植,采用数字表法随机分为4组:0.9%(质量分数)氯化钠注射液对照检查组7只。分别给予①尼美舒利(nimesulide,NIM)+5-氮杂脱氧胞苷(5-Aza-deoxycytidine,5-Aza)干预 ②尼美舒利干预 ③5-Aza干预。5周后处死动物,比较各组移植瘤的直径,计算移植瘤体积,绘制生长曲线。反转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)测定组织中COX-2 mRNA表达;免疫组织化学方法测定COX-2蛋白的表达;亚硫酸氢盐测序方法测定移植瘤中COX-2启动子的甲基化状态。酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)法检测各组移植瘤中前列腺素E2(prostaglandin E2,PGE₂)的浓度。结果各组动物体质量增长差异无统计学意义,组间具有可比性。联合干预组移植瘤明显小于其他各组,差异具有统计学意义(P<0.01)。COX-2 mRNA和蛋白表达结果一致,均表现为联合干预组最低,5-Aza干预组最高。甲基化测序结果10个CpG位点中联合干预组和5-Aza干预组的甲基化率低于尼美舒利组和对照组(30% vs 58.3%)。PGE₂的浓度分析显示联合干预组明显低于对照组和单独尼美舒利干预组,PGE₂比值在联合干预组、尼美舒利干预组分别为0.37、0.91,联合干预组的比值与对照组比较,差异具有统计学意义(P<0.05)。结论低表达COX-2的ESCC移植模型中COX-2启动子甲基化是调节该基因表达的重要机制之一。联合选择性COX-2抑制剂尼美舒利和去甲基化药物5-氮杂脱氧胞苷对低表达COX-2的ESCC移植瘤的生长有协同抑制作用。

关键词: 食管鳞癌, 环氧合酶-2, DNA甲基化, 移植瘤

Abstract:

Objective Overexpression of cyclooxygenase-2(COX-2) is known to be associated with the carcinogenesis of esophageal squamous cell carcinoma(ESCC), but in some ESCC cell lines the expression level of COX-2 may be low. In this study, methylation state of cyclooxygenase-2 gene promoter in ESCC was examined in xenografts to investigate the relationship between promoter methylation and COX-2 expression. We also studied the effect of COX-2 selective inhibitor nimesulide in conjunction with 5-aza-2 deoxycytidine on the growth of xenografts. Methods KYSE150 cell line with low COX-2 expression was used to establish ESCC xenograft in nude mice. The mice were divided into four groups, nimesulide(NIM)+5-Aza-DC(5-Aza) (group 1), NIM group(group 2), 5-Aza group(group 3), and sodium chloride control group(group 4). The growth of those xenografts in nude mice was observed. Methylation state of cyclooxygenase-2 gene promoter in xenografts was monitored using bisulfate sequencing method. RT-PCR and immunohistochemistry were employed to determine the expression level of COX-2 mRNA and protein in xenograft samples, respectively. PGE₂ concentration in xenografts was measured by ELISA. Results During the trial, mice grew well and no infection or death was observed. No difference of weight was found between groups(P>0.05). Xenograft volumes were different between groups with that of the sodium chloride group smaller than other groups, and the difference was significant(P<0.01). It was showed that methylation degree of COX-2 gene promoter was higher in NIM group and sodium choloride group than that in NIM combining 5-Aza group and 5-Aza group. For 10 CpG sites, methylation degrees were 30% and 58.3% in group 3 and group 4, respectively. The expressions of COX-2 mRNA and protein were coincidently low in group 1 and high in group 3. PGE₂ values were 0.37, 0.91 and 1.22 in group 1, 2 and 3 compared with group 4, respectively. Conclusion Promoter methylation of COX-2 gene is one of the important mechanisms to regulate COX-2 expression in low COX-2 expression ESCC xenografts. Combination of selective COX-2 inhibitor and 5-Aza has synergistic effect on ESCC xenografts.

Key words: esophageal squamous cell carcinoma, cyclooxygenase-2, DNA methylation, xenografts

中图分类号:

R735.1

王青釭, 孟莹, 朱圣韬, 施海韵, 张澍田. 启动子甲基化调节在低表达环氧合酶-2的食管鳞癌移植模型中的作用[J]. 首都医科大学学报, 2014, 35(6): 798-804.

Wang Qinggang, Meng Ying, Zhu Shengtao, Shi Haiyun, Zhang Shutian. Effect of promoter methylation on lower expressed cyclooxygenase-2 esophageal squamous cell carcinoma xenograft[J]. Journal of Capital Medical University, 2014, 35(6): 798-804.

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启动子甲基化调节在低表达环氧合酶-2的食管鳞癌移植模型中的作用

Effect of promoter methylation on lower expressed cyclooxygenase-2 esophageal squamous cell carcinoma xenograft

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