CAJ | 학술논문

目的建立HRM技术定量检测DLC-1启动子甲基化的方法 ,并分析探讨DLC-1甲基化程度与前列腺癌病理参数的相关性.方法选取89份前列腺癌组织以及10份匹配癌旁组织标本,采用LCM收集肿瘤细胞群,抽提DNA后进行甲基化修饰.以CpGenome Universal Methylated DNA作为100%甲基化样本,以100%非甲基化的健康人外周血DNA作为稀释剂,分别制成100%、80%、50%、30%、10%、0%系列浓度的标准曲线,并进行重复性和灵敏性评价.同时用HRM技术定量检测前列腺癌细胞中DLC-1甲基化水平,探讨DLC-1甲基化程度与前列腺癌患者年龄、PSA水平与前列腺癌TNM的关系.结果 100%、80%、50%、30%、10%、0%甲基化标准品的HRM熔解曲线从右往左依次排列,10份癌旁组织标本、35份前列腺癌组织标本重叠在0%标准曲线上;5份前列腺癌组织标本位于0%～30%区域内,29份位于31%～80%区域内,20份位于81%～100%区域内.HRM技术最低检测限可达10%的甲基化水平,优于MSP检测(30%甲基化水平).DLC-1甲基化水平与前列腺癌患者年龄无明显相关(X~2=3.29,P=0.19),与PSA水平也无相关性(X~2=2.04,P=0.36),但DLC-1甲基化水平与TNM分期显著相关(X~2=9.04,P=0.01),且随TNM分期增高而增高.结论成功建立的HRM技术定量检测DLC-1甲基化水平的方法稳定、灵敏、操作简单,DLC-1启动子甲基化有望成为评估前列腺癌TNM的分子指标.
목적 건립HRM기술정량검측DLC-1계동자갑기화적방법 ,병분석탐토DLC-1갑기화정도여전렬선암병리삼수적상관성.방법 선취89빈전렬선암조직이급10빈필배암방조직표본,채용LCM수집종류세포군,추제DNA후진행갑기화수식.이CpGenome Universal Methylated DNA작위100%갑기화양본,이100%비갑기화적건강인외주혈DNA작위희석제,분별제성100%、80%、50%、30%、10%、0%계렬농도적표준곡선,병진행중복성화령민성평개.동시용HRM기술정량검측전렬선암세포중DLC-1갑기화수평,탐토DLC-1갑기화정도여전렬선암환자년령、PSA수평여전렬선암TNM적관계.결과 100%、80%、50%、30%、10%、0%갑기화표준품적HRM용해곡선종우왕좌의차배렬,10빈암방조직표본、35빈전렬선암조직표본중첩재0%표준곡선상;5빈전렬선암조직표본위우0%～30%구역내,29빈위우31%～80%구역내,20빈위우81%～100%구역내.HRM기술최저검측한가체10%적갑기화수평,우우MSP검측(30%갑기화수평).DLC-1갑기화수평여전렬선암환자년령무명현상관(X~2=3.29,P=0.19),여PSA수평야무상관성(X~2=2.04,P=0.36),단DLC-1갑기화수평여TNM분기현저상관(X~2=9.04,P=0.01),차수TNM분기증고이증고.결론 성공건립적HRM기술정량검측DLC-1갑기화수평적방법 은정、령민、조작간단,DLC-1계동자갑기화유망성위평고전렬선암TNM적분자지표.
Objective To establish quantitative method for detection of methylation level of DLC-1 promoter with HRM technology to analyze its association with pathological parameters in prostate cancer.Methods 89 prostate cancer tissue samples and 10 matched normal tissue samples were enrolled into this study.Prostate cancer cells were obtained by LCM.DNA was extracted and modified for methylation determination.CpGenome Universal Methylated DNA was chosen as 100% methylation sample.Then the calibrators representative of 100%,80%,50%,30%,10% and 0% methylation levels were prepared with dilution in a DNA sample of peripheral blood from healthy subjects(100% non-methylation).The DLC-1 methylation levels in prostate cancer tissue samples were detected with HRM.The associations of methylated level with age of patients,PSA value,TNM stage were investigated respectively.ResultsThe melting curves representing 100%,80%,50%,30%,10% and 0% methylation levels were aligned from right to left.The methylation levels of 10 adjacent normal samples and 35 prostate cancer samples were overlapped with 0% methylated calibrator.The methylation levels of 5 cancer samples ranged between 0% and 30%.The methylation levels of 29 cancer samples ranged between 31% and 80%.The methylation levels of 20 cancer samples ranged between 81% and 100%.HRM could be used to reliably detect the as low as 10% methylation for each assay,whereas methylation specific PCR(MSP) could be used to detect 30% methylation level.No significant association between methylation level and patients' age(X~2=3.29,P=0.19),PSA level(X~2=2.04,P=0.36) was found.However,DLC-1 methylation was higher in the prostate cancer tissues with advanced TNM stage(X~2=9.04,P=.01).Conclusions The quantitative method for DLC-1 methylation with HRM is successfully established.It is convenient with good reproducibility and high sensitivity.DLC-1 methylation could be used as the molecular marker for estimation of malignancy in prostate cancer.