喉癌组织中LCRG1基因启动子的克隆、突变和甲基化检测  

Cloning,mutation and methylation detection of human LCRG1 gene promoter in laryngeal squamous cell carcinoma

作　　者：李金花[1,2] 曾龙武[2] 谢海龙[2] 

机构地区：[1]杭州师范大学附属医院病理科,杭州310015 [2]南华大学肿瘤研究所,衡阳421000

出　　处：《临床与实验病理学杂志》2013年第8期819-824,共6页Chinese Journal of Clinical and Experimental Pathology

基　　金：国家自然科学基金(81172576)

摘　　要：目的观察喉癌候选抑瘤基因(laryngeal carcinoma related gene 1,LCRG1)启动子的功能。方法 (1)通过5'-缺失突变、瞬时转染与荧光素酶报告基因活性检测,进一步分析与LCRG1基因表达调控有关的最小启动子区域;(2)通过PCR-单链构象多态性(PCR-single-strand conformation polymorphism,PCR-SSCP)技术检测40例喉癌组织中LCRG1基因启动子突变;(3)应用甲基化特异性PCR(methylation specific PCR,MSP)技术检测40例喉癌组织中LCRG1基因启动子甲基化状态;(4)采用RTPCR技术检测40例喉癌组织中LCRG1基因的表达。结果 (1)LCRG1基因最小启动子片段位于转录起始位点上游-169^-57位点;(2)喉癌组织中LCRG1基因启动子无突变;(3)40例喉癌组织中有5.00%LCRG1基因启动子甲基化改变;(4)RTPCR检测显示40%(16/40)喉癌组织中LCRG1基因表达下调。结论 LCRG1基因最小启动子位于转录起始位点上游-169^-57位点的DNA片段;LCRG1基因启动子甲基化部分改变可能参与该基因的表达调控。Purpose To investigate the promoter function of laryngeal carcinoma related gene 1 （ LCRG1 ） in laryngeal carcinoma. Methods The 5 ＇-deletion mutagenesis, transient transfection and the luciferase reporter gene were used to analyze the minimal promoter; PCR-single strand conformation polymorphism （SSCP） to detect the mutation in the promoter sequence of the LCRG1 gene in 40 laryngeal carcinoma patients; methylation specific PCR （MSP） to analyze the Methylation of LCRG1 gene promoter CpG island; and semi-quantitive reverse transcription PCR （RT-PCR） to measure the expression of LCRG1 in laryngeal carcinoma. Results The minimal promoter of LCRG1 gene was defined to a fragment from -169 to -57 position relative to the transcription start site. No mutation was found in the promoter sequence of the LCRG1 gene in laryngeal carcinoma; methylation of LCRG1 gene promoter was detected in 5.00% laryngeal carcinoma; and LCRG1 gene was significantly down-regulated in 16 of 40 （40%） primary laryngeal carcinomas. Conclusions The minimal promoter of LCRG1 gene is defined to a fragment from - 169 to - 57 position relative to the transcription start site. Methylation of LCRG1 gene promoter may be involved in the regulatory mechanisms of LCRG1 gene expression.

关 键 词：转录调控 最小启动子 瞬时转染 甲基化 

分 类 号：R739.65[医药卫生—肿瘤]