摘要
目的探究正常结直肠黏膜、结直肠腺瘤和结直肠腺癌(colorectal cancer,CRC)的Cp G岛甲基化表型(CIMP)差异及相关分子病理机制。方法随机选取中国人民解放军火箭军特色医学中心送检结直肠组织160例,按照组织病理改变分为正常黏膜组、低级别腺瘤组、高级别腺瘤组、CRC组,每组40例,采用甲基化实时荧光定量PCR(Methylight)法检测CIMP相关基因甲基化状态;使用高通量测序法("Next-generation"sequencing technology,NGS)检测CRC中62个肿瘤易感基因的突变负荷;利用真核表达载体技术过表达CRC细胞LOVO的甲基化转移酶3 A(DNMT3A),采用实时荧光定量PCR(Quantitative Real-time PCR,q PCR)及免疫组化技术检测DNMT3A、Cyclin D1、p16的基因及蛋白表达。结果正常黏膜组的CIMP-0占比为50%(20/40例),CIMP-L占比50%(20/40例),无CIMP-H;低级别腺瘤组中CIMP-0为32.5%(13/40例),CIMP-L为67.5%(27/40例),无CIMP-H;高级别腺瘤组中CIMP-0占12.5%(5/40例),CIMP-L占75%(30/40例),CIMP-H占12.5%(5/40例);CRC组中CIMP-0占5%(2/40例),CIMPL占77.5%(31/40例),CIMP-H占17.5%(7/40例)。CRC组中CIMP相关基因甲基化发生数量和肿瘤易感基因的突变数量呈正相关,线性回归方程R=0.130,P=0.022;过表达DNMT3A后,SW620细胞DNMT3A mRNA及蛋白表达量明显上升(P<0.001),p16基因发生甲基化,p16基因mRNA及蛋白表达量下降(P<0.05),Cyclin D1基因甲基化状态未发生变化,仍为非甲基化状态,但Cyclin D1基因及蛋白表达量上升(P<0.001)。结论①启动子区Cp G岛甲基化是导致肿瘤发生的重要机制之一,其可能通过甲基化使肿瘤易感基因序列的稳定性下降,导致更易发生致癌突变。②DNA甲基转移酶3A过表达可引起肠癌细胞CIMP表型改变并继而导致相关基因的沉默或活化。
Objective To explore the differences in CpG island methylation phenotype(CIMP)and related molecular pathological mechanisms in normal colorectal mucosa,colorectal adenoma,and colorectal cancer(CRC).Methods A total of 160 colorectal tissues were randomly selected from the Chinese People’s Liberation Army Rocket Army Medical Center for this study.They were divided into normal mucosa group,low-grade adenoma group,high-grade adenoma group,and CRC group according to the histopathological changes.Detection of methylation status of CpG island methylation phenotype(CIMP)related genes was carried out by methylation real-time quantitative PCR(Methylight);mutation load of 62 tumor susceptibility genes was detected in colorectal cancer using NGS high-throughput sequencing.Eukaryotic expression vector technology was used to overexpress DNA methyltransferase 3A(DNMT3A)in colorectal cancer cells LOVO,and real-time fluorescent quantitative PCR(qPCR)and immunohistochemical techniques were used to detect the gene and protein expression of DNMT3A,Cyclin D1,and P16.Results The proportion of CIMP-0 in normal mucosa group was 50%(20/40 cases),and the proportion of CIMP-L was 50%(20/40 cases).There was no CIMP-H;CIMP-0 was 32.5%(13/40 cases)in the low-grade adenoma group.CIMP-L was 67.5%(27/40 cases),without CIMP-H;CIMP-0 accounted for 12.5%(5/40 cases),and CIMP-L accounted for 75 in the high-grade adenoma group%(30/40 cases);CIMP-H accounted for 12.5%(5/40 cases);CIMP-0 accounted for 5%(2/40 cases)and CIMP-L accounted for 77.5%(31/40 cases)in the colon cancer group CIMP-H accounted for 17.5%(7/40 cases).In the colorectal adenocarcinoma group,there was a positive correlation between the number of methylation of CIMP-related genes and the number of mutations in tumor-susceptible genes.Linear regression equation was R=0.130,P=0.022.After overexpression of DNMT3A,the expression of DNMT3A mRNA and protein in SW620 cells was significantly increased(P<0.001),but P16 gene methylation,P16 gene mRNA and protein expression decreased(P<0.05);cy
作者
罗宇
孙锁柱
刘赛娜
程波
刘珊
许琳
LUO Yu;SUN Suo-zhu;LIU Sai-na;CHENG Bo;LIU Shan;XU Lin;无(Institute of Basic Research Chengde Medical University,Chengde,Hebei 067000,China;Department of Pathology,Chinese People's Liberation Army Rocket Force Special Medical Center,Beijing 100088,China;Hebei North University,Zhangjiakou,Hebei 075000,China)
出处
《诊断病理学杂志》
2020年第10期697-701,705,共6页
Chinese Journal of Diagnostic Pathology
关键词
结直肠癌
CPG岛甲基化
CpG岛甲基化表型
基因突变
病理机制
Colorectal cancer
CpG island methylation
CpG island methylation phenotype
Gene mutation
Pathological mechanism
