DNMT3a表达上调与A549细胞对顺铂耐受关系的实验探究

The Up-regulation Expression of DNMT3a Resensitizes A549 Cell to Cisplatin

基因甲基化状态改变是肿瘤获得性耐药产生的重要途径之一。DNMTs是基因甲基化的重要调节因子。为了寻找预测肺腺癌A549细胞对顺铂敏感性的生物标志,该研究以A549细胞和对顺铂耐受的同源A549细胞(A549-DDP)为研究对象,利用质粒转染技术上调A549细胞DNMT3a表达,通过RT-PCR检测转染前后DNMT3a在mRNA水平的表达情况。在不同浓度顺铂作用下,通过MTT法、克隆形成实验检测转染前后细胞增殖率并计算IC50和计数细胞克隆形成数。在相同顺铂浓度作用下,流式细胞仪检测转染前后细胞凋亡分数的改变。结果显示:顺铂作用24 h后,转染DNMT3a表达质粒的A549细胞对顺铂IC50明显高于A549细胞[(9.19±0.91)μmol/L vs(4.96±0.58)μmol/L,P=0.000],转染后的细胞凋亡率明显低于未转染细胞[(1.33±0.38)%vs(5.22±0.67)%,P=0.039];不同浓度顺铂连续作用5 d后,转染DNMT3a表达质粒的A549细胞形成的克隆数均明显高于A549细胞组。结果提示,DNMT3a表达上调是肺腺癌A549细胞对顺铂获得性耐受的重要原因之一。检测肺癌患者血清DNMT3a水平作为预测肺癌患者对顺铂敏感性的生物标志值得进一步研究。

The change of gene methylation status is a main cause for acquired chemo resistance of cancer. DNMTs plays an indispensable role in methylation regulations. In order to find a biomarker predicting chemo sensitivity of A549 cell to cisplatin, human lung adenocarcinoma A549 cells were chosen and transfected with a DNMT3a plasmid to enhance the expression of DNMT3a. Semi-quantitative RT-PCR was performed to confirm the expression ofDNMT3a mRNA before and after plasmid transfect. Exposing to different concentrations of cispaltin, MTT assays were used to evaluate cell viability and calculate the IC50 value to cisplatin before and after plasmid transfect respectively; clone formation assay were detected to evaluate the clone forming ability before and after cell transfect. Exposing to the same concentration of cisplatin, flow cytometric analysis was used to assess apoptosis rate before and after plasmid transfect. The results indicated： exposing to cisplatin 24 h, IC50 value to cisplatin increased greatly in A549 cell with exogenous expression of DNMT3a than that in A549 cell [（9.19±0.91） μmol/L vs （4.96±0.58）μmol/L, P=0.000]. After exposing to cisplatin for 5 days, the clone number of A549 with exogenous expression of DNMT3a was significantly increased than that ofA549; with the same concentration of cisplatin, the apoptosis percentage decreased greatly in A549 with exogenous expression of DNMT3a than that ofA549 [（1.33±0.38）% vs （5.22±0.67）%, P=0.039]. The results suggest that the up-regulation expression of DNMT3a may be a vital cause for acquired chemo resistance of lung cancer and it needs deeply study about serum DNMT3a acts as biomarker and a predictive factor for chemosensitivity of lung cancer patients in clinic.