摘要
目的:探讨食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)组织中蛋白酪氨酸磷酸酶1(protein tyrosin phosphatase 1,SHP-1)基因异常低表达的表观遗传学调节机制及其临床意义。方法:所用组织标本均来自河北医科大学第四医院2008-2011年行食管癌根治术并且病理诊断为ESCC患者的癌组织及相应癌旁组织(距癌灶边缘2 cm以上),共71例。ESCC细胞株(Eca109、Kyse170、Yes-2)培养完成后,进行甲基化抑制剂5-Aza-dC或组蛋白去乙酰化酶抑制剂TSA处理,qPCR和Western blotting实验检测ESCC组织和细胞系中SHP-1 mRNA和蛋白的表达变化,亚硫酸氢盐基因组测序(bisulfite genome sequencing,BGS)法检测ESCC细胞系中SHP-1基因启动子区CpG位点的甲基化频率,甲基化特异性PCR(methylation specific PCR,MSP)技术检测ESCC组织和细胞中SHP-1启动子区的甲基化状态,应用双荧光素酶报告基因实验检测SHP-1启动子区CpG岛甲基化对其转录活性影响。分析ESCC组织中SHP-1甲基化状态分别与临床病理特征和SHP-1 mRNA表达的关系,对组织SHP-1甲基化水平与ESCC患者生存率进行Kaplan-Meier生存分析和Log-Rank检验。结果:5-Aza-dC处理后,SHP-1 mRNA蛋白在3种细胞株中的表达显著上调(均P<0.05),同时其启动子区的甲基化程度均明显降低(均P<0.05);应用TSA处理细胞株后,SHP-1在各细胞株中的表达情况及甲基化状态无明显改变(P>0.05);甲基转移酶处理细胞荧光素报告载体活性显著低于未处理细胞荧光素报告载体活性(P<0.05),表明SHP-1的甲基化可抑制自身的转录。ESCC组织中启动子区的甲基化率明显高于癌旁组织(P<0.05),并与TNM分期、病理分级及淋巴结转移密切有关(P<0.05);与癌旁组织相比,ESCC组织中SHP-1 mRNA相对表达量显著降低(P<0.05),并与启动子区甲基化有关(P<0.05);Kaplan-Meier分析显示,启动子区高甲基化与ESCC患者的不良预后有关(P<0.05)。结论:ESCC组织和细胞株中SHP-1基因启动
Objective:This study aimed at investigating the epigenetic regulation mechanism of abnormally low expression of SHP-1 gene in esophageal squamous cell carcinoma(ESCC).Methods:A total of 71 cases of ESCC tissues and corresponding para-cancer tissues(2 cm from the edge of the cancer)resected during surgery at the Department of thoracic surgery of Hebei Province,the Fourth Hospital of Hebei Medical University from 2008 to 2011 were collected for this study.The expression level of SHP-1 mRNA and protein was detected in esophageal cancer cell lines(Eca109,Kyse170,Yes-2)before and after 5-Aza-dC or TSA treatment by RT-qPCR and Western blotting methods respectively.The methylation status of CpG sites in promoter region of SHP-1 was analyzed by bisulfite genome sequencing(BGS)in three esophageal cancer cell lines before and after 5-Aza-dC treatment.The methylation status of SHP-1 was studied by methylation-specific polymerase chain reaction(MSP)method in esophageal cancer cell lines,ESCC tissues and para-cancer tissues.The association between the SHP-1 promoter methylation status and clinic pathological parameters were analyzed in ESCC patients.Dual-luciferase reporter assay systems method was applied to detect the impacts of methylation status of CpG island in SHP-1 promoter region on gene transcription activity.For prognostic analysis of SHP-1 methylation,survival curves were constructed using the Kaplan-Meier method and the log-rank.Results:After treated with 5-Aza-dC,the expression level of SHP-1 mRNA and protein was significantly up-regulated in Eca109,Kyse170 and Yes-2 cells,meanwhile the methylation status of SHP-1 was decreased(P<0.05).The expression level of SHP-1 had no obviously change after treated with trichostatin A(TSA).The methylation frequency of promoter in ESCC tumor tissues was significantly higher than that in corresponding para-cancer tissues(P<0.05).When stratified for clinic pathologic characteristics,methylation frequency of SHP-1 was associated with TNM stage,pathological differentiation,and LN
作者
刘磊
张少为
刘新波
刘俊峰
LIU Lei;ZHANG Shaowei;LIU Xinbo;LIU Junfeng(Department of Thoracic Surgery,the Fourth Hospital of Hebei Medical University,Shijiazhuang 050011,Hebei,China)
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
北大核心
2020年第6期602-608,共7页
Chinese Journal of Cancer Biotherapy
基金
国家自然科学基金资助项目(No.81572441)
河北省医学科学研究重点课题资助项目(No.20180588,20170700)。
关键词
食管鳞状细胞癌
蛋白酪氨酸磷酸酶1基因
启动子
甲基化
预后评估
esophageal squamous cell carcinoma(ESCC)
protein tyrosin phosphatase 1(SHP-1)
promoter
methylation
prognostic evaluation
