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RASSF1A表达下降可能是肺癌对顺铂获得性耐药的主要因素之一。为了寻找预测顺铂对肺腺癌A549细胞化疗敏感性的生物标志,该研究以对顺铂耐药的同源A549细胞(A549-DDP)为研究对象,利用转染技术上调RASSF1A表达。RT-PCR和Western blot检测转染前后RASSF1A在mRNA和蛋白水平的表达情况。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活力并计算IC50和计数克隆形成数。在同一浓度顺铂作用下,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RASSF1A表达质粒的A549-DDP细胞对顺铂IC50明显低于A549-DDP细胞[(39.9±6.3)μmol/L vs(53.0±5.8)μmol/L,P=0.036];不同浓度顺铂连续作用5天后,转染RASS-F1A表达质粒的A549-DDP细胞形成的克隆数明显少于A549-DDP细胞组;在同一浓度顺铂作用下,转染RASSF1A表达质粒的A549-DDP凋亡分数明显大于A549-DDP细胞[(7.10±0.01)%vs(3.80±0.18)%,P=0.002]。结果提示,RASSF1A表达下降可能是肺腺癌A549细胞对顺铂获得性耐药的重要因素之一,RASSF1A表达在临床上作为肺癌患者顺铂化疗耐受的生物标志值得进一步研究。
The decrease of RASSF1A expression may be one of the main factors of acquired resistance to cisplatin in lung cancer. In order to find a biomarker for predicting the chemosensitivity of cisplatin to human lung adenocarcinoma A549 cells, A549-DDP cells were used to investigate the expression of RASSF1A in A549 cells. The mRNA and protein expression of RASSF1A before and after transfection were detected by RT-PCR and Western blot. Under different concentrations of cisplatin, cell viability was assayed by MTT assay and colony formation assay, and IC50 was calculated and the number of clones formed was counted. Under the same concentration of cisplatin, the change of apoptosis score before and after transfection was detected by flow cytometry. The results showed that IC50 of A549-DDP cells transfected with RASSF1A expression plasmid was significantly lower than that of A549-DDP cells [(39.9 ± 6.3) μmol / L vs (53.0 ± 5.8) μmol / L, P = 0.036]. After treated with different concentrations of cisplatin for 5 days, A549-DDP cells transfected with RASS-F1A expression plasmid had significantly fewer colonies than A549-DDP cells. Under the same concentration of cisplatin, RASSF1A The apoptosis index of A549-DDP expressing plasmid was significantly higher than that of A549-DDP cells [(7.10 ± 0.01)% vs (3.80 ± 0.18)%, P = 0.002]. The results suggest that the decreased expression of RASSF1A may be one of the important factors of acquired resistance to cisplatin in lung adenocarcinoma A549 cells. RASSF1A expression is clinically useful as a biomarker of cisplatin chemoresistance in lung cancer patients.