The Relationship Between Abnormal Expression Of DNMT1, DNMT3a And The Sensitivity To Cisplatin Among Lung Adenocarcinoma Cells

Objective: The change of gene methylation status is a main cause foracquired chemo resistance of cancer. DNMT1, DNMT3a plays anindispensable role in methylation regulations. Previous reportsdemonstrated that the alterations of DNMT1, DNMT3a expression might beassociated with the regulation of chemo sensitivity in cancer. Thus, presentstudy aims to investigate the relationship between DNMT1, DNMT3a andthe sensitivity to cisplatin among A549/A549-DDP cell lines by means ofcell plasmids transfection and shRNA interference. We hope to lay thefoundation for the further study on the resistance mechanism of cisplatinamong lung cancer, to find the predictive biomarkers for clinicalindividualized chemotherapy of lung cancer.Methods: Human lung adenocarcinoma A549cells were chosen andtransfected with LZRS-DNMT1, pcDNA3/Myc-DNMT3a plasmids toenhance the expression of DNMT, DNMT3a. Semi-quantitative RT-PCR and Western-blot analysis were performed to confirm the expression levelsof DNMT1, DNMT3a mRNA and protein before and after plasmidstransfected. The methylation status of RASSF1A was determined bymethylation specific PCR(MSP), Western-blot was used to evaluate theexpression levels of RASSF1A. Exposing to different concentrations ofcisplatin (0-10mol/L), MTT assays were used to evaluate cell viability andcalculate the IC50value to cisplatin before and after plasmids transfectedrespectively; clone formation assay were detected in cisplatin (0,2.5,5μmol/L)to evaluate the clone formation ability before and after celltransfecting. Exposing to the same concentration of cisplatin (2.5μmol/L)for24h, flow cytometric analysis were used to assess apoptosis rate beforeand after plasmid transfected.The eukaryotic expression vector carrying RNAi sequence targetingconserved domain of DNMT1, DNMT3a mRNA and negative control (HK)in pGensil-1were constructed by using technology of gene recombinationand were transfected in A549-DDP cell line. Semi-quantitative RT-PCRand Western-blot analysis were performed to confirm the expression levelsof DNMT1, DNMT3a mRNA and protein before and after plasmidstransfected in24h,48h,72h. Exposing to different concentrations ofcisplatin (0-80μmol/L), MTT assays were used to evaluate cell viability andcalculate the IC50value to cisplatin before and after plasmids transfectedrespectively; clone formation assay were detected in cisplatin（0,25, 50μmol/L) to evaluate the clone formation ability before and after celltransfecting. Exposing to the same concentration of cisplatin (25μmol/L)for24h, flow cytometric analysis were used to assess apoptosis rate beforeand after plasmids transfected.Results: RT-PCR and Western-blot indicated that DNMT1, DNMT3amRNA and protein levels were increased greatly in A549cell transfectedwith LZRS-DNMT1, pcDNA3/Myc-DNMT3a. MSP showed thatRASSF1A was hypermethylated and low expressed in plasmids transfectedcells. After exposing to cisplatin for24h, IC50value to cisplatin increasedgreatly in A549cell with exogenous expression of DNMT1, DNMT3a thanthat in A549cell [(9.14±0.59)μmol/L vs (5.49±1.00)μmol/L, P=0.001；(9.19±0.91) μmol/L vs (4.96±0.58) μmol/L, P=0.000]. After exposing tocisplatin for5days, the clone numbers of A549with exogenous expressionof DNMT1, DNMT3a were significantly increased than that of A549; withthe same concentration of cisplatin, the apoptosis rate decreased greatly inA549with exogenous expression of DNMT1, DNMT3a than that ofA549[(2.24±0.36)%vs (4.43±1.03)%, P=0.036,(1.33±0.38)%vs(5.22±0.67)%, P=0.039].After transfecting with pGenesil-1-DNMT1-shRNA, pGenesil-1-DNMT3a–shRNA plasmids, RT-PCR and Western-blot indicated thatDNMT1, DNMT3a mRNA and protein levels decreased greatly inA549-DDP cell. Exposing to cisplatin for24h, IC50value to cisplatin decreased greatly in A549-DDP cell with down-regulation of DNMT1,DNMT3a than that in A549-DDP cell [(46.13±5.15)μmol/L vs(64.79±12.96)μmol/L, P=0.004;(46.63±7.48)μmol/L vs (67.50±13.80)μmol/L, P=0.001]. After exposing to cisplatin for5days, the clone numbersof A549-DDP with down-regulation of DNMT1, DNMT3a weresignificantly decreased than that of A549-DDP; with the sameconcentration of cisplatin, the apoptosis rate increased greatly inA549-DDP with down-regulation of DNMT1, DNMT3a than that ofA549-DDP [(21.99±5.80)%vs (10.95±1.51)%, P=0.033;(21.44±3.81)%vs(10.95±1.51)%, P=0.02].Conclusion: The abnormal expression of DNMT1, DNMT3a mayplay a critical role in regulating chemo-sensitivity for lung cancer, theclinical value of detecting the expression levels of DNMT1, DNMT3a inNSCLC might provide potential biomarkers for predicting the sensitivity oflung cancer patients to cisplatin.