The Correlation Between Methylation Of DNA Repair Genes And Drug Resistance Of Lung Cancer A549Cells In Cisplatin Chemotherapy

objective Dispose of non-small cell lung carcinomaA549cell line andits cisplatin (DDP) A549/DDP resistant strains with demethylation reagent5-nitrogenimpurity-2’-deoxy cytidine(5-Aza-CdR), check the expression and methylation statusof hMLH1, MGMT genes of lung carcinoma A549cells and A549/DDP cells beforeand after the intervene with demethylation reagent5-Aza-CdR.5-Aza-CdR andcisplatin plus application, IC50and the change of apoptosis in A549/DDP cells.To investigate the correlation between promoter methylation of DNA repair geneshMLH1, MGMT and cisplatin resistance in lung carcinoma A549cells. Methods Theexperiment was divided into non-small cell lung carcinoma A549cells group andA549/DDP cells group. After processing with different concentration of5-Aza-CdR(0,5,10and20mu mol/L), the methylation status of hMLHl, MGMT gene in eachgroup is checked with methylation specific PCR (MSP). hMLH1, MGMT genemRNA expression in each group is detected with real-time fluorescent quantitativePCR. Tetramethyl azo thiazole blue method was used to detect the absorbance valueof A549cells, A549/DDP cells and to methylation of A549/DDP cells treating withdemethylation reagent after48h treatment with different concentration of DDP,observation cell inhibition rate; apoptosis rate of each cell group was determined byflow cytometry. Results (1) hMLHl is the unmethylated status and MGMT is partlyunmethylated in lung cancer A549cells. HMLH1and MGMT are partly methylatedin A549/DDP cells.hMLHl and MGMT gene demethylation in10mu mol/L5-Aza-CdR or20mu mol/L5-Aza-CdR intervened in A549/DDP cells.（2）Usingfluorescence quantitative PCR determination, we found hMLHl, MGMT genemRNAs were expressed in the A549and A549/DDP cells. The hMLHl, MGMTgene mRNA expression of A549/DDP cells was significantly lower than the A549 cells （P<0.05）. When treated with5μmol/L of5-Aza-CdR, the HMLH1mRNAand MGMT mRNA expression of A549/DDP cells were lower than that of A549cellswith a statistically significance（P<0.05）. However, there was no statisticallysignificant difference in10mu mol/L of5-Aza-Cde intervened hMLH1mRNAexpression of A549/DDP group compared with A549cells group（P>0.05）. Thedifference was statistically significant in MGMT mRNA expression of A549/DDPgroup compared with A549cell group (P <0.05); In20mu (mol/L5-Aza-Cdeintervened MGMT gene mRNA expression of A549/DDP and A549cell group hasno statistically significant difference（P>0.05）.(3)When using MTT method todetect cell proliferation in the A549/DDP group, A549Group and the A549/DDPgroup treated with20μmol/L5-Aza-CdR, we found that the IC50value by differentconcentrations cisplatin treatment for48hours concentration of3.50mu mol/L,24.45mu mol/L,10.72mu mol/L, respectively, but the difference was statisticallysignificant between them（P<0.05）. It suggests that A549/DDP cells sensitivity tocisplatin was enhanced by20mu mol/L of5-Aza-CdR treatment; but compared withA549cells, it is still lower.(4) To detect cell apoptosis using flow cytometry，Whenwe added5,10and40mu mol/L DDP for a48-hour treatmet，the apoptosis rate ofA549/DDP group cultured with20mu mol/L of5-Aza-CdR was16.81％、26.09％、51.91％，and the cell apoptosis rate was7.26%,7.26%,15.53%, respectively. Thedifference was statistically significant between the two groups(P<0.05). Therefore itsuggests an enhanced5-Aza-CdR demethylation of cisplatin chemotherapy sensitivityin A549/DDP cells. Conclusion (1)The gene methylation of hMLH1、MGMTpromoter may be a reason of cisplatin resistance to platinum-based chemotherapy insmall cell lung cancer A549cell line.(2)Demethylation drug5-Aza-CdR can inhibitehMLHl、MGMT gene promoter demethylation，restore the mRNA expression ofhMLHl、MGMT，and enhance cell sensitivity to cisplatin. There is a significantcertain correlation among them in the A549/DDP cell line．...