Study Of DNA Methyltransferase And Methylation Status Of Four Tumor Suppressor P16, RASSF1A, CDH1, CDH13 In Lung Carcinoma

Objective1. To study the significances of methylation status in the four tumor suppressor gene (TSG) concluding p16, RASSF1A, CDH13, CDH1 in early detection for lung cancer.2. To detect the transcription level and catalytic activity of DNA methyltransferase ( DNMTs ) and methylation status of the four TSG, and observe the proliferation status of lung cancer cell lines A549 exposed in 5-Aza-CdR ( 5-aza-2'- deoxycitydine ).Methods:1. We selected 77 patients who were suspected suffering from lung cancer, and collected their BALF ( bronchoalveolar lavage fluid) .We used MSP(Methylation- specific PCR)to detect the four TSG's methylation status. 77 patients were divided into 3 groups according to the results of some examinations such as bronchoscopy. First one was lung cancer group. The second one was innocence lung disease group. The third one was follow-up group. The methylation status of the four TSG in the 3 groups was analyzed.3. A549 cells were exposed in the 5-Aza-CdR for demethylation. DNMTs and the four TSG's mRNA levels were determined by semi-quantitative RT-PCR. The catalytic activity of DNMTs was detected by enzyme colorimetric determination kit. The apoposis and changes of cell cycles were observed by flow cytometry ( FCM ), and MTT assay was used to study the cell proliferation. Results1. The lung cancer group included 47 patients. The positive rate of the four TSG's methylation status in lung cancer group was higher than that in other two groups. No significant difference was found between the positive rates of squamous carcinoma and adenocarcinoma in lung cancer group.2. The percentage of methylation that occurred at least one of the four TSG in lung cancer group was 79%. The percentages of methylation that occurred at least one of the three ones in any combination of three were respectively 62 %, 72%, 68%, 72%. There was no significant difference in the positive rates between three-gene combinations and the four-gene combination.3. CDH13 showed partly demethylation in A549 cells after being exposed to 5-Aza-CdR. The transcription levels of the TSG became higher except p16 after demethylation.4. No significant difference in the quantity of DNMTs mRNA can be observed after demethylation, Catalytic activity of DNMTs decreased after demethylation. FCM recommended 5-Aza-CdR arrested A549 cell cycle at G1/G0. The rate of apoptosis in drug group was higher than the control group. Cell proliferation of drug group was inhibited according to the results of MTT assays.ConclusionThere is relationship between the occurrence of lung cancer and the methylation status of p16, RASSF1A, CDH1 and CDH13 in BALF. Any three ones'combination from the four TSG has diagnosis significance in lung cancer. 5-Aza-CdR has cytostatic agent in A549 cells cultured in vitro, and accelerates its apoptosis and enhances the transcription levels of RASSF1A,CDH1,CDH13. The inhibition of DNMTs catalytic activity can account for demethylating agent of 5-Aza-CdR in A549 cells, not the DNMTs transcription level.