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中国癌症防治杂志

中国癌症防治杂志 ›› 2019, Vol. 11 ›› Issue (1): 30-36.doi: 10.3969/j.issn.1674-5671.2019.01.06

• 结直肠癌专栏 • 上一篇    下一篇

E2F1基因过表达对人结肠癌耐奥沙利铂细胞株HCT116/L多药耐药性的影响

  

  1. 广西医科大学附属肿瘤医院胃肠外科
  • 出版日期:2019-02-25 发布日期:2019-03-28
  • 通讯作者: 唐卫中 E-mail：2354531686@qq.co
  • 基金资助:
    国家自然科学基金项目（81502120；81560454）

Effects of over-expression E2F1 gene on multi-drug resistance of human colon cancer Oxaliplatin-resistant cell line HCT116/L

  • Online:2019-02-25 Published:2019-03-28

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摘要： 目的 探讨E2F1基因过表达对人结肠癌耐奥沙利铂细胞株HCT116/L多药耐药性的影响及作用机制。方法 HCT116/L细胞分为HCT116/L+E2F1组、HCT116/L+NC组和HCT116/L组，HCT116/L+E2F1组和HCT116/L+NC组分别转染E2F1过表达重组慢病毒载体（pCMA-E2F1-HA）及其阴性对照慢病毒载体（pCMA-HA）。Western blot检测E2F1蛋白的表达水平；MTT法检测各组对化疗药物（阿霉素、5-FU和顺铂）的敏感性；流式细胞仪检测各组细胞内阿霉素的泵出率和细胞凋亡情况；细胞划痕实验检测细胞迁移情况。Western blot进一步检测耐药相关基因MDR1蛋白的表达水平。结果 HCT116/L+E2F1组的E2F1蛋白表达水平明显高于HCT116/L+NC组和HCT116/L组（P＜0.05）；HCT116/L+E2F1组对阿霉素、5-FU和顺铂的IC50值分别为（1.61±0.21） μg/mL﹑（5.22±0.12） μg/mL﹑（3.52±0.15） μg/mL，高于HCT116/L+NC组的（0.61±0.11） μg/mL、（3.93±0.54） μg/mL、（2.31±0.45） μg/mL和HCT116/L组的（0.69±0.13） μg/mL、（4.19±0.51） μg/mL、（2.51±0.42） μg/mL（P＜0.05）；HCT116/L+E2F1组阿霉素的泵出率为（22.51±0.12）%，亦高于HCT116/L+NC组的（10.92±0.09）%和HCT116/L组的（8.45±0.11）%（P＜0.05）；HCT116/L+E2F1组细胞凋亡率为（6.42±0.52）%，低于HCT116/L+NC组的（12.81±0.69）%和HCT116/L组的（11.45±0.31）%（P＜0.05），HCT116/L+E2F1组能增强细胞迁移能力（P＜0.05），并阻滞细胞于S期。HCT116/L+E2F1组中MDR1蛋白表达量为0.41±0.08，高于HCT116/L+NC组的0.19±0.08和HCT116/L组的0.15±0.07（P＜0.05）。结论 E2F1 基因过表达可降低人结肠癌耐奥沙利铂细胞HCT116/L 对化疗药物的敏感性，上调MDR1基因表达，使化疗药物在结肠癌细胞内的蓄积浓度降低，从而增强人结肠癌耐奥沙利铂细胞HCT116/L的多药耐药性。

关键词: 结肠癌, E2F1, 多药耐药性, 阿霉素, 5-FU, 顺铂, MDR1

Abstract: Objective To investigate the effects of over-expression E2F1 gene on multi-drug resistance of human colon cancer Oxaliplatin-resistant cell line HCT116/L. Methods HCT116/L cells were divided into 3 groups（HCT116/L+E2F1 group，HCT116/L+NC group and HCT116/L group）. HCT116/L+E2F1 group cells were infected with an over-expression E2F1 recombinated lentiviral vector，HCT116/L+NC group cells were infected with an empty lentiviral vector，and HCT116/L group with no treatment . The expression levels of E2F1 protein was detected by Western blot analysis. The sensitivities to chemotherapeutic drugs（Adriamycin，5-FU，and Cisplatium） were assessed using MTT. The pump-out accumulation rate of Adriamycin and the apoptosis of cells were detected using flow cytometry. The migration of cells was dected by cell scratch test. Western blot was used to detect the expression level of resistance-related gene MDR1. Results The expression level of E2F1 in HCT116/L+E2F1 group was higher than that in HCT116/L+NC group and HCT116/L group（P＜0.05）. The IC50 of Adriamycin，5-FU，and Cisplatium of HCT116/L cells in HCT116/L+E2F1 group were（1.61±0.21） μg/mL，（5.22±0.12） μg/mL and （3.52±0.15） μg/mL，respectively，which were significantly higher than that in HCT116/L+NC group［（0.61±0.11） μg/mL，（3.93±0.54） μg/mL，（2.31±0.45） μg/mL] and HCT116/L group［（0.69±0.13） μg/mL，（4.19±0.51） μg/mL，（2.51±0.42） μg/mL］. The pump rate of Adriamycin was （22.51±0.12）% in HCT116/L+E2F1 group，while HCT116/L group was （8.45±0.11）% and HCT116/L+NC group was（10.92±0.09）%，and the differences were statistically significant（P＜0.05）. The apoptosis rate of HCT116/L+E2F1 group was （6.42±0.52）%，which was significantly lower than that of HCT116/L+NC group （12.81±0.69）% and HCT116/L group （11.45±0.31）%. HCT116/L+E2F1 group could enhance cell migration ability （P<0.05） and arrested cells in S phase. The expression of MDR1 protein in the HCT116/L+E2F1 group was 0.41±0.08，which was significantly higher than 0.19±0.08 in the HCT116/L+NC group and 0.15±0.07 in the HCT116/L group. Conclusions Over-expression of E2F1 gene can reduce the sensitivity of human colon cancer oxaliplatin-resistant HCT116/L to chemotherapeutic drugs and up-regulate the expression of MDR1 gene，which reduce the accumulation of chemotherapeutic drugs in HCT116/L cells，then enhance the multi-drug resistance of HCT116/L cells.

Key words: Colorectal cancer, E2F1, Multi-drug resistance, Adriamycin, 5-FU, Cisplatium, MDR1

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