启动子DNA甲基化和组蛋白乙酰化对胃癌中PDX1表达的影响  

DNA Methylation and Histone Acetylation of the Promoter Regulate PDX1 Expression in Gastric Cancer

作　　者：马娟[1] 余莲英[1] 廖山婴[1] 王蓓蓓[1] 徐丽姝[1] 沙卫红[1] 王启仪[1] 刘庆华[2] 

机构地区：[1]广东省人民医院//广东省医学科学院//广东省老年医学研究所消化科,广东广州510080 [2]中山大学附属第一医院肾内科,广东广州510080

出　　处：《中山大学学报（医学科学版）》2016年第2期175-182,共8页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(81001112);广州市科技局珠江科技新星项目(2012J2200019);广东省医学科研基金(A2014021)

摘　　要：【目的】已知胃癌中PDX1表达下调,本研究旨在探讨DNA甲基化和组蛋白乙酰化修饰对PDX1基因表达的调控。【方法】免疫组化法检测胃癌组织芯片中PDX1、Dnmt1和HDAC的表达,分析其相关性。5’-aza-d C和TSA处理胃癌细胞,RT-PCR检测PDX1 m RNA水平,分析DNA去甲基化和组蛋白乙酰化对PDX1表达的影响;构建6个PDX1启动子片段至p GL3载体,检测转录活性,分析去甲基化和乙酰化对PDX1启动子转录活性的影响。MSP和BSP评估PDX1启动子DNA甲基化状态以及启动子活性受5’-aza-d C和Sss I的影响;Ch IP评估PDX1启动子组蛋白乙酰化状态以及启动子活性受TSA的影响。【结果】免疫组化结果显示与正常组织对比,胃癌组织中的PDX1表达下调,Dnmt1和HDAC表达上调,PDX1和Dnmt1及HDAC的表达负相关(P<0.05);5’-aza-d C和TSA使PDX1在胃癌细胞中重获表达;F383是PDX1启动子核心,5’-aza-d C和TSA增强F383转录活性,Sss I抑制F383转录活性;MSP结果显示F383在胃癌细胞中呈完全甲基化;BSP结果显示与对照比较,位于F383区域内17个Cp G位点中80%以上呈现甲基化。Ch IP分析结果提示F383呈组蛋白H3和H4低乙酰化。【结论】PDX1基因在胃癌中表达沉默与启动子DNA高甲基化和组蛋白低乙酰化有关。[Objective] The expression of pancreatic-duodenal homeobox 1 （PDX1） is aberrantly reduced in gastric cancer. This study aimed to determine the regulation of DNA methylation and histone acetylation on the expression of PDX1 in gastric cancer. [ Methods] The expression of PDX1, Dnmtl and HDAC was detected in human gastric cancer tissue chips by immunohistochemistry, and its correlations were analyzed. PDX1 expression in response to demethylation （5＇-aza-dC） and acetylation （TSA） was assessed in human gastric cancer cells by reverse transcription-polymerase chain reaction （RT-PCR）. Six PDX1 promoter fragments were cloned into pGL3 vector and the transcription activity of promoters in response to demethylation and acetylation was detected by dual luciferase assay. In gastric cancer cell lines, promoter DNA methylation was evaluated by methylation-specific PCR （MSP） and bisulfite DNA sequencing PCR （BSP） while histone acetylation was detected by chromatin immunoprecipitation （CHIP） assay. [Results] Compared to gastric normal tissues, the expression of PDX1 protein was weak but the expression of Dnmtl and HDAC was upregulated. Our results also showed that PDX1 is negatively correlated with Dnmtl and HDAC. PDX1 expression was restored by 5＇ - aza-dC and by TSA in gastric cancer cells. F383 （-22063 - -21681nt） wihich displayed significant promoter transcript activity, which was increased by 5＇-aza-dC and TSA but decreased by SssI methylase. F383 was completely methylated in gastric cancer cells by MSP and more than 80% of 17 single CpG sites located at F383 were methylated compared with gastric normal tissues by BSP （P 〈 0.01 ）. F383 had also lower acetylation level of histone H3 and H4 in gastric cancer cells by ChIP assay. [Conclusion] PDX1 silencing was resulted by promoter hyper-methylation and histone hypo-acetylation in gastric cancer.

关 键 词：胃癌 PDX1 DNA甲基化 组蛋白乙酰化 

分 类 号：R73[医药卫生—肿瘤]