肺癌细胞株中EPS8表达下降对顺铂化疗敏感性的影响

摘要

目的 探究肺癌细胞株中表皮生长因子受体通路底物8(EPS8)表达下降对顺铂化疗敏感性的影响.方法 脂质体转染法将EPS8沉默后并通过实时荧光定量聚合酶链反应(qRT-PCR)检测EPS8在各细胞系的沉默效率.采用Caspase-3/7细胞凋亡实验检测,5μmol顺铂对淋巴母细胞样细胞系(LCLs)和A549肺癌细胞凋亡的影响.Alamar Blue细胞生长抑制实验中采用0.01μmol/L光神霉素A作为EPS8抑制剂单用及联用不同浓度顺铂,Alamar Blue细胞生长抑制实验检测其对癌和非癌细胞系生长抑制的影响.结果 与对照组比较,LCLs细胞系中EPS8的沉默导致顺铂干预时细胞存活率升高7.9%,凋亡率下降8.7%.与此相反,顺铂干预导致肺癌细胞存活率下降20.6%.光神霉素A在LCLs细胞系中能够降低EPS8的表达,并与对照组比较,非癌细胞系LCLs在顺铂干预时具有更低Caspase-3/7活性,凋亡率降低.在5种非小细胞肺癌(NSCLC)细胞系中,光神霉素A同样能够导致EPS8表达下降.与LCLs细胞系比较,光神霉素A的干预能够使4种NSCLC细胞和膀胱癌细胞HTB9对顺铂的敏感性增加(<0.05),但NSCLC中存在EGFR突变的H1975细胞对顺铂的敏感性并未出现统计学改变(>0.05).结论 通过核糖核酸(RNA)干扰或者光神霉素A将EPS8表达抑制后,肿瘤细胞对顺铂的敏感性升高,而正常细胞LCLs对其敏感性却出现下降.对EPS8分子机制的进一步研究有望其成为抗肿瘤治疗的新靶点.%Objective To evaluate the role of epidermal growth factor receptor pathway substrate 8 (EPS8) in cellular susceptibility to cisplatin in tumor cells and normal cells. Methods EPS8 RNA interference was used to determine EPS8 silencing efficiency in each cell line by qRT-PCR. Caspase-3/7 was used to detect the apoptosis of lymphoblastoid cell lines (LCLs) and A549 lung cancer cell when treated with 5 μmol/L cisplatin after EPS8 was silenced. In the Alamar Blue cell growth inhibition assay, 0.01 μmol/L mycophenolate as an EPS8 inhibitor was used alone or in combination with different concentrations of cisplatin to detected its effect on the growth inhibition of cancer and non-cancer cell lines. Results Compared with the control group, the survival rate of EPS8 in LCLs cell line was increased by 7.9% and the apoptosis rate decreased by 8.7%. In contrast, cisplatin intervention resulted in a 20.6% reduction in lung cancer cell survival. The expression of EPS8 was decreased in LCLs cell line, and the Caspase-3/7 activity was lower in the non-cancer cell line LCLs treated by cisplatin compared with the control group, and the apoptosis rate was decreased. In the five kinds of non-small cell lung cancer (NSCLC) cell lines, mithramycin also lead to decreased expression of EPS8. Compared with the LCLs cell line, the sensitivity to cisplatin increased significantly in the four NSCLC cells and the bladder cancer cell HTB9 ( <0.05), but it had no statistically significant in the NSCLC, the EGFR mutated H1975 cells ( >0.05). Conclusions There are significantly increased of sensitivity to cisplatin in cancer cells when EPS8 inhibition through the RNA interference or mithramycin treated,while normal cells decrease its sensitivity.