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中华肝脏外科手术学电子杂志

中华肝脏外科手术学电子杂志 ›› 2016, Vol. 05 ›› Issue (02) : 124 -128. doi: 10.3877/cma.j.issn.2095-3232.2016.02.014

所属专题： 文献；

基础研究

siRNA沉默MRP5基因对人肝癌细胞HepG2多药耐药性的影响
杨尚霖1, 刘建平1,†(), 刘波2, 苏正1, 韦金星1, 张克韬1   
  1. 1. 510120　广州，中山大学孙逸仙纪念医院胆胰外科
    2. 510530　广州，中山大学附属第三医院岭南医院普通外科
  • 收稿日期:2015-11-11 出版日期:2016-04-10
  • 通信作者: 刘建平
  • 基金资助:
    广东省科技计划项目(2011B031800296); 广州市产学研专项(1561000198)

Effect of MRP5 gene silenced by siRNA on multi-drug resistance of human hepatocellular carcinoma cells HepG2

Shanglin Yang1, Jianping Liu1,†(), Bo Liu2, Zheng Su1, Jinxing Wei1, Ketao Zhang1   

  1. 1. Department of Hepatobiliary and Pancreatic Surgery, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China
    2. Department of General Surgery, Lingnan Hospital, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510530, China
  • Received:2015-11-11 Published:2016-04-10
  • Corresponding author: Jianping Liu
  • About author:
    Corresponding author: Liu Jianping, Email:
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引用本文：

杨尚霖, 刘建平, 刘波, 苏正, 韦金星, 张克韬. siRNA沉默MRP5基因对人肝癌细胞HepG2多药耐药性的影响[J/OL]. 中华肝脏外科手术学电子杂志, 2016, 05(02): 124-128.

Shanglin Yang, Jianping Liu, Bo Liu, Zheng Su, Jinxing Wei, Ketao Zhang. Effect of MRP5 gene silenced by siRNA on multi-drug resistance of human hepatocellular carcinoma cells HepG2[J/OL]. Chinese Journal of Hepatic Surgery(Electronic Edition), 2016, 05(02): 124-128.

目的

探讨siRNA沉默多药耐药相关蛋白（MRP）5基因对人肝癌耐药细胞多药耐药性的影响。

方法

构建人肝癌耐药细胞HepG2/表柔比星（ADM），合成MRP5-siRNA片段，使用Lipofectamine 2000脂质体转染细胞。实验分3组，siRNA组为HepG2/ADM转染MRP5-siRNA，耐药组为HepG2/ADM细胞，普通组为肝癌细胞HepG2。MTT法检测细胞对化疗药物的敏感性，实时荧光定量RT-PCR检测MRP5基因mRNA相对表达量，Western blot法检测MRP5蛋白表达，流式细胞术检测细胞凋亡情况。多组比较采用单因素方差分析，两组间比较采用LSD-t检验或t检验。

结果

siRNA组细胞对ADM、氟尿嘧啶（5-FU）和奥沙利铂（OXA）的半抑制浓度（IC50）分别为（0.180±0.008）、（1.657±0.014）、（0.055±0.007）mg/L，耐药组相应为（0.317±0.035）、（3.785±0.523）、（0.129±0.009）mg/L，普通组相应为（0.022±0.008）、（0.163±0.010）、（0.080±0.012）mg/L，耐药组对3种化疗药物的IC50明显高于普通组（t=14.202，11.993，13.937；P<0.05），siRNA组明显低于耐药组（LSD-t= -6.609，-7.044，-11.257；P<0.05）。siRNA组、耐药组、普通组细胞的MRP5 mRNA相对表达量分别为1.377±0.141、3.858±0.481、1.000±0.374，耐药组较普通组明显升高（LSD-t=9.600，P<0.05），siRNA组较耐药组明显降低（LSD-t=-11.669，P<0.05）。siRNA组、耐药组、普通组细胞MRP5蛋白灰度值分别为816、2 245、58，耐药组较普通组明显增强，siRNA组较耐药组明显减弱。siRNA组细胞凋亡率为（25.1±3.7）%，明显高于耐药组的（3.3±0.7）%（t=9.950，P<0.05）。

结论

MRP5与人肝癌细胞的多药耐药性相关，siRNA沉默MRP5基因可以上调肝癌细胞对化疗药物的敏感性。

关键词: 癌，肝细胞, 多药耐药相关蛋白质类, 抗药性，肿瘤, 核糖核酸酶类
Objective

To investigate the effect of multidrug resistance-associated protein (MRP) 5 silenced by small interfering ribonucleic acid (siRNA) on the multi-drug resistance of human hepatocellular carcinoma (HCC) cells.

Methods

Multi-drug resistance human HCC cells HepG2/epirubicin(ADM) were constructed and MRP5-siRNA fragment was synthesized. The cells were transfected with Lipofectamine 2000. The experiment was divided into three groups. HepG2/ADM cells transfected with MRP5-siRNA were allocated in the siRNA group, HepG2/ADM cells in the drug resistance group and HCC cells HepG2 in the common group. The cellular sensitivity to chemotherapy agents was detected by MTT assay. The relative expression of MRP5 messenger ribonucleic acid (mRNA) was detected by real-time fluorescence quantification RT-PCR. The expression of MRP5 protein was detected by Western blot. Cellular apoptosis was detected by flow cytometry. Multiple groups were compared by one-way ANOVA and two groups were compared by LSD-t test or t test.

Results

The half-inhibitory concentration (IC50) of cells for ADM, fluorouracil (5-FU) and oxaliplatin (OXA) in the drug resistance group was (0.317±0.035), (3.785±0.523) and (0.129±0.009) mg/L, significantly higher compared with (0.022±0.008), (0.163±0.010) and (0.080±0.012) mg/L in the common group (t=14.202, 11.993, 13.937; P<0.05). The IC50 for the three chemotherapy drugs in the siRNA group was (0.180±0.008), (1.657±0.014) and (0.055±0.007) mg/L, significantly lower than those in the drug resistance group (LSD-t=-6.609, -7.044, -11.257; P<0.05). The relative expression level of MRP5 mRNA in the drug resistance group was 3.858±0.481, significantly higher compared with 1.000±0.374 in the common group (LSD-t=9.600, P<0.05). The relative expression level of MRP5 mRNA in the siRNA group was 1.377±0.141, significantly lower than that in the drug resistance group (LSD-t=-11.669, P<0.05). The gray value of MRP5 protein was 2 245 in the drug resistance group, significantly up-regulated compared with 58 in the common group. The gray value of MRP5 protein was 816 in the siRNA group, significantly down-regulated compared with that in the drug resistance group. The cell apoptosis rate in the siRNA group was (25.1±3.7)%, significantly higher compared with (3.3±0.7)% in the drug resistance group (t=9.950, P<0.05).

Conclusions

MRP5 is associated with the multi-drug resistance of human HCC cells. MRP5 gene silenced by siRNA can enhance the sensitivity of HCC cells towards chemotherapy drugs.

Key words: Carcinoma, hepatocellular, Multidrug resistance-associated proteins, Drug resistance, neoplasm, Ribonucleases
表1 三组细胞对三种化疗药物的半抑制浓度（mg/L，）
组别 IC50
ADM 5-FU OXA
siRNA组 0.180±0.008 1.657±0.014 0.055±0.007
耐药组 0.317±0.035 3.785±0.523 0.129±0.009
普通组 0.022±0.008 0.163±0.010 0.080±0.012
图1 Western blot法检测三组细胞MRP5蛋白表达情况
图2 流式细胞仪检测两组细胞凋亡情况
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