Methylation of p16 Gene in Plasma and Tissues from Non-small Cell Lung Cancer Patients, Demethylation and the Biological Behavior of Lung Cancer Cell and Transcription of p16 Gene
|School||Anhui Medical University,|
|Course||Biochemistry and Molecular Biology|
|Keywords||p16gene methylation non- small cell lung cancer DNA methylation 5-Aza-CdR DNA demethylation|
ObjectiveTo detect methylation of p16 gene in plasma and tissues from NSCLC patients, and to evaluate the clinical significance. To investigate the effects of 5-aza-2’-deoxycytidine （5-Aza-CdR） on methylation and expression of p16 gene in the human lung cancer cell line A549, and to discuss the mechanism of p16 gene silencing in human lung cancer cells, as well as the regulating effect of the demethylating agent on p16 gene expression.Methods The methylation of p16 gene in DNA from 120 non-small cell lung cancer tissues and corresponding nomalignant tissues were tested with the methods of the methylation-specific PCR（MSP）, and Methylation of p16 gene was tested by MSP among the plasma from 120 cases of NSCLC and the plasma from 120 cases of controls. A549 cells were cultured RPMI 1640 medium and were treated with different concentrations (5×10-6 mol/L, 1×10-6 mol/L, 5×10-7 mol/L, 1×10-7 mol/L) of DNA methyltransferase inhibitor 5-Aza-CdR. Methylation-specific polymerase chain reaction （MSP） was used to detect the promoter methylation state of the p16 gene. RT-PCR were used to detect expression of p16 mRNA with 5-Aza-CdR, respectively.Activation in cell growth was observed by MTT assay and colony formation on plate.Results The total frequency of p16 methylation was 44.2% in lung cancer tissues, significantly higher than that in the nomalignant tissues 11.7%（P<0.01）; 32.5（39/120）of the plasma from 120 cases of NSCLC showed hypermethylation for p16 gene, whereas no methylated p16 gene were found in plasma from the norml controls（P<0.05）. The methylation of p16 gene in DNA did not exhibit the obvious difference between the cancer tissues and the plasma of NSCLC（P>0.05）. The methylation of p16 gene in DNA exhibited the obvious difference between the nomalignant tissues and the plasma of NSCLC（P<0.01）. The frequency of p16 Methylation in female patients was 22.22%, which was 48.04% in male patients.There was not significantly different between them （P> 0.05）; In the age group in 60 years old patients with methylation the detection rate was 53.85%, higher than in patients below 60 years of age the detection rate of 32.73%, compared with a significant difference （P <0.05）. A549 cells were treated with different doses of 5-Aza-CdR displayed a slowed groth rate significantly in contrast to the control group, colony formation was significantly reduced, p16 gene promoter methylation by 5-Aza-CdR treatment, demethylation of the promoter region.Conclusion Detection of the methylation of p16 gene in plasma and lung cancer tissues from NSCLC patients might offer an effective means for the earlier diagnosis of the malignancy. Promoter hypermethylation is a major mechanism of p16 gene silencing in human lung cancer cells, and can be reversed by the demethylating agent 5-Aza-CdR. Demethylating agents can regulate the expression of the p16 gene.