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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。
Gene methylation status change is one of the important pathways of tumor-acquired drug resistance. DNMTs are important regulators of gene methylation. In order to find a biomarker that predicts the sensitivity of lung adenocarcinoma A549 cells to cisplatin, A549 cells and A549-DDP cells that are resistant to cisplatin were selected as research objects. A549 cells were transfected by plasmid transfection DNMT3a expression was detected by RT-PCR DNMT3a mRNA expression before and after transfection. Under the action of cisplatin of different concentrations, the proliferation rate of cells before and after transfection was measured by MTT assay and colony formation assay, and the IC50 and the number of cell clone formation were counted. Under the same cisplatin concentration, the changes of apoptosis score before and after transfection were detected by flow cytometry. The results showed that the IC50 of A549 cells transfected with DNMT3a plasmid was significantly higher than that of A549 cells ([(9.19 ± 0.91) μmol / L vs (4.96 ± 0.58) μmol / L, P = 0.000] (1.33 ± 0.38)% vs (5.22 ± 0.67)%, P = 0.039]. After treated with different concentrations of cisplatin for 5 days, the expression of DNMT3a in transfected cells was significantly lower than that in untransfected cells The number of colonies of A549 cells was significantly higher than that of A549 cells. The results suggest that DNMT3a upregulation of lung adenocarcinoma A549 cells one of the important reasons for the acquired resistance to cisplatin. Detection of serum DNMT3a levels in patients with lung cancer as a biomarker for predicting susceptibility to cisplatin in lung cancer patients deserves further study.