Analysis Of Methylation Status And Gene Expression Of Adenomatous Polyposis Coli (APC) Gene In Lung Cancer Cell Lines And The Threshold Of Different Detection Methods Of APC Gene Methylation With The Simulation Plasma Of Lung Cancer Patients

BackgroundsIn the last decade or so, the use of the word epigenetics has rapidly spread. Identefied epigenetic processes involved in human disease include chromatin remodeling, DNA methylation, and noncoding RNAs regulation. It has been proved that epigenetic events play an important role in many cancers as well as the genetic events.In cancer development, overall methylation is decreased but de novo methylation of some promoter-associated CpG islands occurs. Methylation is the third mechanism besides the mutation and deletion in the inactivation of the tumor suppressor gene. Thus, there is considerable potential for using methylation as a tumor-specific marker.Lung cancer is a major public health problem in the world. The survival rates for lung cancers have changed little over past two decates. A major factor in the high mortality of lung cancer patients is the presence of metastases in approximately two-thirds of patients at the time diagnosis. It has been estimated that detection of lung cancer at early stages could potentially increase survival rates. Thus, investigations into identifying biomarkers are strongly needed for early diagnosis of lung cancer.ObjectiveTo analyze the methylation status and transcription ofAPC gene in 3 lung cancer cell lines and the effect of demethylation in the APC expression. To analyze the threshold of different detection methods of APC gene with the simulation plasma of lung cancer patients and the significance of the methylation detection method to the early diagnosis of lung cancer.Materials and MethodsThe methylation status of the APC gene promoter 1A was analyzed by methylation specific PCR (MSP), microarray and bisulfite sequencing. The expression of APC was examined by real time quantitative polymerase chain reaction with SYBR Green I staining and western blot. NCI-H460 ceils were treated with different concentrates of DNA methyltransferase inhibitor 5-aza-2-deoxycytidine (5-aza-dC), and the expression of APC was examined by real time quantitative polymerase chain reaction with SYBR Green I staining and western blot. DNA in NCI-H460 cell was extracted by the method of phenol/chloroform, then it was quantified and added into the plasma isolated from health people with the decuple dilution density to make simulation lung cancer patients plasma. The plasma DNA was extracted by Chemagic Viral DNA/RNA kit. After bisulfite modification, threshold of both common MSP and real-time quantitative MSP with SYBR Green I and TaqMan probes were analyzed.ResultsIn this study, methylated APC promoter was found in a non-small-cell lung cancer cell line NCI-H460 but unmethylated in small cell lung cancer cell line NCI-H446 and lung adenocarcinoma cell line SPCA1. Monoallelic methylation and methylated 20 CpG islands in APC were found. Expression of the APC in the NCI-H460 cell line declined obviously compared to the other two cell lines. APC expression of NCI-H460 cell line was obviously enhanced after treatment of different concentrations of 5-aza-dC. Real-time quantitative MSP with TaqMan probe and SYBR Green I staining could detect the amount of methylated APC DNA releasing from at least 30 tumor cells/mL and 300 tumor cells/mL while common MSP could give position result only if the number of NCI-H460 cell up to 3000/mL in blood. The sensitivity of the real-time quantitative MSP with TaqMan probe was at least 100 times higher than that of common MSRConclusionsExpression of APC gene was regulated by CpG island methylation. Monoalletic expression ofAPC gene might play an important role in lung cancer. APC expression of NCI-H460 cell line was obviously enhanced after treatment of 5-aza-dC. Real-time quantitative MSP has a higher sensitivity than common MSP. The real-time quantitative MSP may be useful in the early diagnosis of lung cancer.