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

Abstract

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