肝母细胞瘤细胞中STAT3异常表达的DNA甲基化分子机制研究  

DNA methylation mechanism of abnormal STAT3 expression in hepatoblastoma cells

作　　者：季春宜[1] 尹强[1] 袁妙贤[1] 陈立健[1] 高红强[1] 盛新仪 谢惟心 Ji Chunyi;Yin Qiang;Yuan Miaoxian;Chen Lijian;Gao Hongqiang;Sheng Xinyi;Xie Weixin(Department of General Surgery,Hunan Children's Hospital,Changsha 410007,China)

机构地区：[1]湖南省儿童医院普外科,长沙410007

出　　处：《中华小儿外科杂志》2021年第10期893-898,共6页Chinese Journal of Pediatric Surgery

基　　金：湖南省卫健委课题项目基金(C2019011)。

摘　　要：目的探讨肝母细胞瘤(hepatoblastoma,HB)肿瘤组织中信号转导与转录激活因子3(signal transducer and activator oftranscription 3,STAT3)高表达的DNA甲基化机制。方法以湖南省儿童医院普外一科于2016年5月至2019年3月收治的20例HB患儿为研究对象,其中男12例,女8例,中位年龄为33个月,年龄范围为3~72个月;3例甲胎蛋白含量<3000μg/L,17例甲胎蛋白含量>3000μg/L;7例为胎儿型HB,7例为胚胎型HB,1例为未分化型HB,5例为混合型HB;按HB的PRETEXT分期系统分期,Ⅰ期3例,Ⅱ期6例,Ⅲ期6例,Ⅳ期5例。手术切取HB肿瘤及癌旁正常组织,肿瘤组织的切除范围参照术前影像学检查结果,并扩大1~2 cm切除,癌旁正常组织需切取离肿瘤边缘至少2 cm的正常组织。将肿瘤组织作为实验组,癌旁正常组织作为对照组。采用实时定量聚合酶链反应(quantificational real-time polymerase chain reaction,qRT-PCR)及蛋白质印迹法检测实验组和对照组中STAT3和TET的表达水平。采用亚硫酸氢盐测序PCR(bisulfite sequencing PCR,BSP)技术检测实验组和对照组中STAT3启动子区DNA甲基化水平。使用染色质免疫共沉淀(chromatin Immunoprecipitation,ChIP)-定量聚合酶链反应(quantitative PCR,qPCR)检测实验组和对照组中STAT3启动子区TET1结合水平,并将实验组中TET1结合水平与STAT3启动子DNA甲基化水平做相关性分析。结果在两组间STAT3的表达水平差异方面,qRT-PCR检测结果显示,实验组与对照组STAT3的表达水平相比(8.43±5.05比1.03±0.41),实验组STAT3的表达水平显著升高,差异具有统计学意义(t=-6.53,P<0.001);蛋白质印迹法检测证实了实验组STAT3表达水平的上调,实验组与对照组相比(0.96±0.17比0.37±0.13),差异具有统计学意义(t=-12.13,P<0.001)。在两组间STAT3启动子DNA甲基化水平的差异方面,BSP检测结果显示,实验组与对照组STAT3启动子区DNA甲基化水平相比(0.52±0.10比0.82±0.06),实验组STAT3启动子区DNA甲基�Objective To explore the DNA methylation mechanism of high STAT3 expression in tumor tissues of children with hepatoblastoma(HB).Methods From May 2016 to March 2019,20 hospitalized HB children were selected as research subjects.There were 12 boys and 8 girls with a median age of 33(3-72)months.Alpha-fetoprotein concentration was<3000μg/L(n=3)and>3000μg/L(n=17).Clinical types of HB were fetal(n=7),embryonic(n=7),undifferentiated(n=1)and mixed(n=5).According to the staging system of HB PRETEXT,the stages wereⅠ(n=3),Ⅱ(n=6),Ⅲ(n=6)andⅣ(n=5).HB tumor and normal tissue adjacent to cancer were surgically removed.Resection scope of tumor tissue was based on the results of preoperative imaging examination and resection was expanded by 1-2 cm.Normal tissue adjacent to cancer was removed from normal tissue with a margin of at least 2 cm.Tumor tissue was selected as experimental group and normal tissue adjacent to cancer as control group.The expression levels of STAT3 and TET in tumor and adjacent normal tissues were detected by quantificational real-time polymerase chain reaction(qRT-PCR)and Western blot.Sulfite sequencing was employed for detecting DNA methylation in STAT3 promoter region in tumor and adjacent normal tissues.TET1 binding level in STAT3 promoter region in tumor and adjacent normal tissues was detected by ChIP-qPCR and correlation analysis conducted between the levels of TET1 binding and DNA methylation of STAT3 promoter in tumor tissues.Results qRT-PCR results indicated that the expression level of STAT3 was significantly higher in experimental group than that in control group(8.43±5.05 vs.1.03±0.41)and the difference was statistically significant(t=-6.53,P<0.001).Western blot confirmed that the expression of STAT3 was up-regulated in experimental group(0.96±0.17 vs.0.37±0.13)and the difference was statistically significant(t=-12.13,P<0.001).BSP results showed that,as compared with control group,DNA methylation level of STAT3 promoter region declined markedly in experimental group(0.52±0.10 vs

关 键 词：肝肿瘤 信号转导与转录激活因子3 TET家族蛋白1 DNA甲基化 

分 类 号：R735.7[医药卫生—肿瘤]