Methylation Of TIMP-3and P16Gene In Plasma And Tissues From Non-small Cell Lung Cancer Patients, Demethylation And The Biological Behavior Of Lung Cancer Cell And Transcription Of TIMP-3Gene

Objective To detect methylation of TIMP-3gene and p16gene in plasma and tissuesfrom NSCLC patients, and to evaluate the clinical significance. Process therepresentative human lung adenocarcinoma cells by different concentrations ofdemethylational drug folic acid(FA). Observe its biological behaviors such asproliferation,differentiation,invasion and metastasis. Detect the methylation status andits mRNA transcription level of the tumor suppressor gene TIMP-3. To provide a basisfor application of folic acid in the clinical treatment of NSCLC.Methods A methylation-specific PCR(MSP) was performed for detecting themethylation of the TIMP-3gene and p16gene in the DNA of the lung cancer tissues,paraneoplastic tissues, and plasma from110NSCLC patients. The determination wasalso conducted on110normal blood samples. The results of the groups were compared.A549cells were cultured RPMI1640medium and were treated with differentconcentrations (15、75、375、1875nmol/L) of DNA methyltransferase inhibitor FA. Usethe MTT assay to detect the A549cell proliferation affected by FAwith differentconcentration and time; Use the cell adhesion assay to detect the A549cell adhesionability affected by FA with different concentration; Use the tablet colony formationassay to detect the A549single-cell clone forming ability affected by FA with differentconcentration; Use the cell migration inhibition assay to detect rhe A549cell migrationaffected by FA with different concentration; Methylation-specific polymerase chainreaction (MSP) was used to detect the promoter methylation state of the TIMP-3gene. RT-PCR were used to detect expression of TIMP-3mRNA with FA.Results The total frequency of the TIMP-3, p16,joint detection methylation separatelywas26.7%、44.2%、59.2%in lung cancer tissues, which was significantly higher thanthose in the nomalignant tissues6.5%、11.7%、14.2%（P<0.01）.The detection rate ofhypermethylation for TIMP-3,p16and joint detection partly was13.3%、32.5%、40%, inthe plasma of the110NSCLC cases, and no methylated gene was found in the plasmaof the control group（P<0.01）.The joint detection rate of hypermethylation in the plasmadid not significantly correlate with the clinical classification, the pathologic types, andclinical staging of the NSCLC. In the concentration range of15-375nmol/L, FA hasinhibited effecting on some biological characteristics of A549cell, such as proliferationactivity, single cell cloning capacity, ability of cell adhesion, migration ability, and has adose-response. But the concentration rose to1875nmol/L, the inhibition of FA reduces.Within15-375nmol/L concentration range, FA demethylate the TIMP-3gene andincrease its transcriptional activity.Conclusion Joint detection for hypermethylation of TIMP-3and p16genes can improvethe positive rate, and provide valuable information for screening and diagnosis of theNSCLC. Promoter hypermethylation is a major mechanism of TIMP-3gene silencing inhuman lung cancer cells, and can be reversed by the demethylating agent FA.Demethylating agents can regulate the expression of theTIMP-3gene. It also provides a basis for FA used in the clinical treatment of NSCLC.