BGC-823人胃癌细胞株SOX2基因的甲基化状态及其去甲基化的意义  

Methylation and demethylation of SOX2 gene promoter in BGC-823 gastric cancer cells

作　　者：魏斌[1] 刘刚[1] 蔡桦立 李庚鹏 黄河庆 罗琪[1] 黄正接[1] 

机构地区：[1]厦门大学附属第一医院胃肠外科厦门市肿瘤中心福建医科大学第一临床医学院福建中医药大学第一临床医学院,361003

出　　处：《中华医学杂志》2016年第32期2548-2553,共6页National Medical Journal of China

基　　金：福建省科技计划重点项目（2014D017）;福建省自然科学基金科技项目（2015J01564,2016J01639）;福建省医学创新课题（2012-CXB-29）;厦门市科技计划项目（3502220134011）

摘　　要：目的观察BGC-823人胃癌细胞株中性别决定相关基因簇2（SOX2）启动子的甲基化状态，探讨SOX2基因去甲基化后对BGC-823细胞的生长和侵袭能力的影响。方法通过甲基化特异性PCR法（MSP）检测DNA甲基转移酶抑制剂5-氮杂-2’-脱氧胞嘧啶（5-Aza—CdR）对BGC-823细胞SOX2启动子甲基化状态的影响，实时荧光定量PCR及蛋白印迹法检测经不同浓度5-Aza—CdR作用前后BGC-823细胞中SOX2的表达情况，四甲基偶氮唑盐（MTT）法检测存活率，Transwell小室法和划痕实验检测侵袭及转移能力变化。建立裸鼠BGC-823胃癌移植瘤模型，40只移植瘤小鼠平均分为2组，腹腔分别注射磷酸缓冲液（PBS）、5-Aza—CdR，通过免疫组化、蛋白质印迹法检测5-Aza—CdR的抑瘤效果。结果5-Aza—CdR逆转了BGC-823细胞SOX2的甲基化状态，SOX2mRNA表达恢复，且恢复表达程度与5-Aza—CdR药物浓度呈正比。BGC-823细胞SOX2mRNA和蛋白的相对表达量低于正常胃黏膜GES．1细胞[（22．80±0．36）和（0．49±0．01）比（20．36±0．45）和（0．91±0．28），均P〈0．05]，经系列浓度（0、1、10μmol／L）5-Aza．CdR处理后BGC-823细胞SOX2mRNA和蛋白的相对表达量相比差异均有统计学意义[（22．99±0．42）和（0．65±0．19）比（21．78±0．41）和（0．73±0．13）比（20．51±0．47）和（0．83±0．14），均P〈0．05]。经不同浓度（1、10、20μmol／L）处理后，BGC-823细胞的增殖速度明显减慢，与浓度为0μmol／L的对照组比较，各处理组细胞存活率差异有统计学意义（P〈0．05），且迁移、侵袭能力减弱（均P〈0．05）。裸鼠移植瘤结果显示：5-Aza—CdR组的最终肿瘤体积、最终瘤重较小[（286．6±37．5）mm^3和（325．2±32．2）mg比（540．7±42．6）mm^3和（694．7±36．1）mg，P〈0．05]，生存期较长[（22．5±1．0）比（18．7-4-1．6）d，P〈0．05]，SOX2蛋白表达量高[（0．96±0Objective To study the significance and effect of methylation status of sex determining region Y-box 2 （SOX2） gene promoter, and to investigate the effect of demethylation on the cell proliferation and invasion in BGC-823 gastric cancer cells. Methods Methylation-specific PCR （MSP） was used to assess the role of 5-aza-2＇-deoxycytidine （5-Aza-CdR） in the methylation of SOX2 promoter in the BGC-823 cell lines treated with different concentration of 5-Aza-CdR. We mapped the expression of SOX2 in the BGC- 823 cell lines by the quantitative real-time PCR （qPCR） and Western blotting before and after treatment of 5-Aza-CdR. The survival of BGC-823 cells were detected by M3T assay. The invasion and migration of BGC-823 cells were investigated by transwell methods, and the migration of BGC-823 cells was also assessed by the scratch assay exposed to 5-Aza-CdR or vehicle control. Model of transplanted tumor on nude mouse were used to study the anticancer effect of 5-Aza-CdR in vivo by qPCR, Western blotting and immunohistochemistry. Results The methyltransferase inhibitor 5-Aza-CdR restored the loss of SOX2 expression in BGC-823 cell lines in a dose-dependent manner. The mRNA and protein expression of SOX2 had significant difference between the gastric cancer tissues and normal gastric mucosa （mRNA levels： 22. 80 ± 0. 36 vs 20. 36 ± 0.45, P 〈 0. 05 ; protein levels ： 0. 49 ± 0. 01 vs 0. 91 ± 0. 28, P 〈 0. 05）. It also had significant difference among the BGC-823 cell lines treated with 5-Aza-CdR of the different concentrations （0, 1 and 10 μmol/L） （mRNA levels： 22. 99 ± 0. 42 vs 21.78 ± 0. 41 vs 20. 51 ± 0.47, P〈0.05; protein levels： 0.65±0.19 vs0.73±0.13 vs0.83±0.14, P〈0.05）. Compared with the control group （5-Aza-CdR concentration of 0 μmol/L）, the survival rates of BGC-823 cell lines were significantly decreased in treatment groups （5-Aza-CdR concentrations of 1, 10 and 20 μmol/L, all P 〈 0. 05 ）. Restored expression of SOX2 in the BGC-823 cell lines in

关 键 词：胃肿瘤 SOX2启动子 5-氮杂-2'-脱氧胞嘧啶 去甲基化 

分 类 号：R735.2[医药卫生—肿瘤]