Methylation quantification of adenomatous polyposis coli(APC) gene promoter in plasma of lung cancer patients
Shi-Yang Pan,1,2,4 Er-Fu Xie,1,2 Yong-Qian Shu,3,4 Li Gao,1,2 Li-Xia Zhang,1,2 Dan Chen,1,2 Jin-Bu Chen,1,2 Wen-Jun Zhao,1,2 Yuan Mu1,2 and Ji-Nan Zhang41. Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, 210029, P.R. China 2. The Key Laboratory for Laboratory Medicine of Jiangsu Province, Nanjing, Jiangsu, 210029, P.R. China 3. The Platform for Molecular Diagnosis and Biotherapy of Graveness Disease of Jiangsu Province, Nanjing, Jiangsu, 210029, P.R. China 4. Department of Oncology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, 210029, P.R. China
Background and Objective: The protein encoded by adenomatous polyposis coli (APC) gene participates in the signaling transduction pathway. Substantial studies have revealed that hypermethylation of APC gene promoter is closely related to the pathogenesis and development of cancer. This study was to develop a real-time quantitative methylation specific PCR (real-time QMSP) method, and detect the methylation of APC gene promoter in plasma of lung cancer patients. Methods: Genomic DNA with methylated APC gene promoter was extracted from the lung cancer cell line NCI-H460 using phenol-chloroform and quantified by spectrophotometric measurements. DNA was added into 200 μL plasma samples of healthy volunteers to make 10-fold serial dilutions. Circulating DNA from simulated plasma samples, 78 lung cancer patients, 31 patients with benign lung diseases and 23 health controls was extracted using magnetic beads and modified by bisulfite. The concentration of cell-free methylated APC gene promoter in the plasma samples was quantified by the external reference method with the standard curve constructed using simulated plasma. Results: The linear range of the real-time QMSP assay was 1.5×102-1.5×105 copies / mL and its lowest detectability was 1.5 ×102 copies per milliliter plasma. Of 78 lung cancer patients, positive methylation of the APC gene promoter was detected in tumor tissues of 40 cases. Among the 40 lung cancer patients, positive methylation of the APC gene promoter was found in the plasma of 19 patients (47.5% ). The concentrations of methylated APC promoter in the 19 lung cancer patients ranged from 1.67×102 to 6.78×103 copies / mL, with a median concentration of 1.67×103 copies / mL. No positive methylation of the APC gene promoter was detected in the plasma of 38 lung cancer patients without APC gene methylation in tissues, 31 benign lung diseases and 23 healthy controls. Conclusions: The newly developed real-time QMSP method allows the quantitative measurement of APC gene promoter methylation in plasma. Hypermethylation of the APC gene promoter in plasma is a potential diagnostic marker for lung cancer diagnosis.
【Fund】： 江苏省卫生厅重大项目科研基金(No.H200707);; 江苏省科技厅社会发展基金(No.BS2007073 No.BM2006113);; 江苏省实验诊断学重点实验室基金(No.XK200731)~~
【CateGory Index】： R734.2
【CateGory Index】： R734.2