| Objective To investigate the Clinical significance of Detection of promoterhypermethylation of DAPK gene in non-small cell lung cancer and the effects of5-Aza-CdR on this process。In the first part of the experiment.Methods In the first partof the experiment，112patients in NSCLC as experiment groups,and normal as controlgroups were enrolled,by using nested methylation-specific polymerase chain reaction（nMSP） to determine the methylation status of the DAPK gene in the DNA of the lungcancer tissues，paraneoplastic tissues，plasma from peripheral blood and112normalblood samples.The results of the groups were compared. In the second part of theexperiment，Human glandular lung cancer cell A549were cultured in DMEM and thentreated with different concentrations of5-Aza-CdR.The inhibitory rates of cellproliferation were detected by MTT assay. The effect of different concentration of5-Aza-CdR on A549cell migration ability was observed by cell scratch test.Themethylation of DAPK gene in the cell line was detected by methylation specificpolymerase chain reaction （MSP）, and the expression of DAPK was detected byRT-PCR.Results The total frequency of the DAPK gene methylation was59.8%inNSCLC tissues, significantly higher than that in the paraneoplastic tissues at8.0%（P＜0.001）. In squamous cell carcinoma, adenocarcinoma and adenosquamous carcinomathe rates of hypermethylation between the lung cancer tissues and the paraneoplastictissues all exhibit significant difference（P＜0.001）.The rate of hypermethylation for DAPK gene was21.4%in the plasma of the112NSCLC cases,and no methylated genein the plasma of the control group （P<0.001）. The rates of hypermethylation in plasmadid not significantly correlate with the clinical classification,pathologic types andclinical staging of the NSCLC.5-Aza-CdR displayed a growth inhibitory effect onA549cell in a dose and time dependent manner after exposure to5-Aza-CdR atdifferent concentrations (1×10-5，1×10-6，1×10-7and1×10-8mol/L) for24,48,72,96and120hours. And after treatment with72hours the1×10-5，1×10-6mol/L groups of A549cells significantly decreased cell migration ability compared to the control group.Themethylation and loss of DAPK mRNA expression were detected in A549cells before5-Aza-CdR treatment.However,the methylation was reversed and DAPK wasre-expressed after5-Aza-CdR treatment with72hours.Conclusion Detection of theDAPK gene methylation in the plasma of NSCLC patients using nMSP may providevaluable information for early screening and diagnosis of the NSCLC.5-Aza-CdR caninhibit the proliferation of A549cells and decrease its migration ability, And improvethe expression and the methylation status of the DAPK gene. |
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