Quantitative Analysis Of DNA Methylation In Gastric Cancer Using Real-Time Fluorescent Quantitative Polymerase Chain Reaction

Object:Gastric cancer generates from the genetic and epigenetic alterations of oncogenes,tumor suppressor genes,DNA repair genes,cell-cycle regulators,and cell adhesion molecules.Methylation of DNA,as a well-known epigenetic change,plays an important role in tumorigenesis of gastric cancer.The detection of aberrant DNA methylation in tumors has important signifcance for exploring the mechanisms of oncogenesis and may provide new molecular assays for cancer detection.Methods for methylation analysis include bisulfite sequencing,MSP,Methylight and so on.All these methods mainly concentrate on qualitative detection,but the quantitative detection is few.Therefor,many investigators are developing new quantitative methods for methylation analysis.The aim of this study was to establish a highly accurate,sensitive,stable,cost-effective and feasible DNA Methylation quantitative method.In order to test the feasibility and clinical usefulness of this method,we detected the quantitative level of p16 and hMLH1 gene Methylation in gastric carcinomas,premalignant lesions and normal control.Methods:The endoscopic biopsy specimens were collected randomly from 42 gastric carcinomas,46 premalignant lesions and 38 normal control inTianjin Medical University General Hospital Department of Digestive Medicine from the period May 2007 to Jul.2007.We performed the SYBR GreenI Real-Time Fluorescent Quantitative Methylation-Specific PCR by using LightCycler(Roche)Real-time quantitative PCR detecting system,detected the quantitative level of p 16 and hMLH1 gene Methylation in gastric carcinomas,premalignant lesions and normal control.Results:The SYBR GreenⅠReal-Time Fluorescent Quantitative PCR was a highly accurate,sensitive,stable,cost-effective and feasible DNA Methylation quantitative method.The minimal limit for the detection of this method was 1.012×10~2 copies/ul. The mean cycle threshold(Ct)of the six times repeating test for the standard substance(10~4～10~7)was 17.03,20.26,23.21 and 26.63 respectively,and the coefficient of variation(CV)between defferent times was 3.85%,3.35%,3.54%and 2.35% respectively.The melting temperature(T_m)for methylated and unmethylated alleles after bisulfite treatment was at 84.0℃～88.0℃,and there was only a peak corresponding to the specific PCR product.There was no specific peak for the negative and blank control group.The Tm value for methylated gene was higher than that for unmethylated gene.The quantitative level for methylated p16 gene in gastric carcinomas, premalignant lesions and normal control was(logarithm)6.99±1.20,5.40±1.48 and 3.55±1.55 respectively.The comparison of the quantitative level for methylated p16 gene among gastric carcinomas,premalignant lesions and normal control had statistically significance(P＜0.05),and the comparison of the quantitative level for any two groups had statistically significance(P＜0.05).In the stomach,the quantitative level for methylated p16 gene increased with histopathologic progression. The quantitative level for unmethylated p16 gene in gastric carcinomas,premalignant lesions and normal control was(logarithm)3.78±1.77,6.58±1.42 and 7.31±1.08 respectively.The comparison of the quantitative level for unmethylated p16 gene among gastric carcinomas,premalignant lesions and normal control had statistically significance(P＜0.05),and the comparison of the quantitative level for any two groups had statistically significance(P＜0.05).In the stomach,the quantitative level for unmethylated p16 gene degraded with histopathologic progression.No statistical significance was observed in gastric carcinomas between the quantitative level of the methylated p16 gene and these clinicopathological characteristics(age,sex,location, tumor histology)(P＞0.05).But there was statistical significance in gastric carcinomas between the quantitative level of the methylated p16 gene and these clinicopathological characteristics(lymphatic metastasis,hematogenous metastasis and the level of malignancy)(P＜0.05).The quantitative level for methylated hMLH1 gene in gastric carcinomas,premalignant lesions and normal control was(logarithm) 7.07±1.50,4.87±1.71 and 2.48±0.51 respectively.The comparison of the quantitative level for methylated hMLH1 gene among gastric carcinomas,premalignant lesions and normal control had statistically significance(P＜0.05),and the comparison of the quantitative level for any two groups had statistically significance(P＜0.05).In the stomach,the quantitative level for methylated hMLH1 gene increased with histopathologic progression.The quantitative level for unmethylated hMLH1 gene in gastric carcinomas,premalignant lesions and normal control was(logarithm) 5.74±1.98,6.47±1.17 and 7.38±1.26 respectively.The comparison of the quantitative level for unmethylated hMLH1 gene among gastric carcinomas,premalignant lesions and normal control had statistically significance(P＜0.05),and the comparison of the quantitative level for any two groups had statistically significance(P＜0.05).In the stomach,the quantitative level for unmethylated hMLH1 gene degraded with histopathologic progression.No statistical significance was observed in gastric carcinomas between the quantitative level of the methylated hMLH1 gene and these clinicopathological characteristics(age,sex,tumor histology and hematogenous metastasis)(P＞0.05).But there was statistical significance in gastric carcinomas between the quantitative level of the methylated hMLH1 gene and these clinicopathological characteristics(location,lymphatic metastasis and the level of malignancy)(P＜0.05).Conclusions:1.We successfully established a novel highly accurate,sensitive,stable, cost-effective and feasible DNA Methylation quantitative method.2.The quantitative level for methylated p16 and the hMLH1 gene increased with gastric mucosa pathological changes.3.There was statistical significance in gastric carcinomas between the quantitative level of the methylated p16 gene and these clinicopathological characteristics (lymphatic metastasis,hematogenous metastasis and the level of malignancy).There was statistical significance in gastric carcinomas between the quantitative level of the methylated hMLH1 gene and these clinicopathological characteristics (location,lymphatic metastasis and the level of malignancy).4.p16 and hMLH1 gene methylation were early events in gastric carcinogenesis,and p16 and hMLH1 gene methylation might be good marks in diagnosis of early gastric cancer.The quantitative detection of p16 and hMLH1 gene methylation had important clinical significance for the prognosis and early monitoring of gastric cancer.