抑制高迁移率族蛋白1表达逆转肝癌细胞Bel-7402/ADM耐药机制研究

Inhibition of HMGB1 expression reverses adriamycin resistance in Bel-7402/ADM human drug resistance hepatocellular carcinoma cells

目的研究高迁移率族蛋白1(HMGB1)对多柔比星(ADM)诱导肝癌细胞凋亡的影响,探讨其在肝癌细胞化疗耐药逆转中的作用。方法建立肝癌细胞耐药株模型Bel-7402/ADM,采用HMGB1小干扰RNA(siRNA)转染肝癌耐药细胞株,四甲基偶氮唑蓝(MTT)法检测转染前后多柔比星对BEL-7402/ADM的半抑制浓度(IC50),流式细胞法检测细胞凋亡情况,采用qRT-PCR检测HMGB1 mRNA表达,蛋白质印迹法检测HMGB1、糖基化终产物受体(RAGE)、核因子-κB(NF-κB)亚单位p-P65和Bcl-2蛋白表达,Caspase活性检测试剂盒检测Caspase-9和Caspase-3活性。采用SPSS 22.0对数据进行统计学分析,多组间差异采用单因素方差分析,两两多重比较采用LSD-t检验。结果HMGB1 siRNA转染后的BEL-7402/ADM细胞HMGB1 mRNA和蛋白表达水平下降,与对照组、Si-NC组相比表达水平差异均具有统计学意义(0.42±0.06 vs 1.00±0.15、1.04±0.22,P=0.004、P=0.003;0.11±0.02 vs 0.33±0.03、0.30±0.02,P<0.001、P<0.001)。与对照组、Si-NC组相比,HMGB1 siRNA组细胞中RAGE(0.39±0.02 vs 0.51±0.03,P=0.014;0.39±0.02 vs 0.51±0.07,P=0.014)、p-P65(0.32±0.04 vs 0.74±0.05,P<0.001;0.32±0.04 vs 0.76±0.06,P<0.001)和Bcl-2(0.50±0.05 vs 1.18±0.09,P<0.001;0.50±0.05 vs 1.06±0.10,P<0.001)蛋白表达下降且差异均具有统计学意义。HMGB1 siRNA组Caspase-9和Caspase-3酶活性高于对照组、Si-NC组且差异均具有统计学意义(3.17±0.38 vs 0.58±0.09,P<0.001;3.17±0.38 vs 0.53±0.11,P<0.001;3.42±0.40 vs 0.68±0.11,P<0.001;3.42±0.40 vs 0.69±0.09,P<0.001)。转染HMGB1 siRNA后,BEL-7402/ADM对多柔比星的敏感性增强,IC50从4.05μg/mL下降至1.04μg/mL;与空白对照组和阴性对照组相比,转染HMGB1 siRNA组多柔比星对BEL-7402/ADM细胞凋亡诱导作用也增强,不同浓度下HMGB1 siRNA组凋亡率与对照组、Si-NC组比较差异均具有统计学意义(17.63±4.52 vs 5.46±0.63,P<0.001;17.63±4.52 vs 5.50±0.61,P<0.001;65.19±5.46 vs 29.73±6.12,P<0.001;65.19±5.46 vs 32.11±4.31,P<0.001)。结论抑制HMGB1表达可以增强BEL-7402/ADM对多柔比星的敏感性,对BEL-7402/ADM耐药性有逆转作用,其机制可能与增强了NF-κB介导的BEL-7402/ADM凋亡有关。

Objective This study aimed to address the alteration of adriamycin(ADM)-induced apoptosis in Bel-7402/ADM human hepatocellular carcinoma cells when high mobility group box-1(HMGB1)expression was inhibited.Methods Bel-7402/ADM with chemoresistance,a human hepatocellular carcinoma cell line,was established and HMGB1-small interference RNA(siRNA)was transfected into the Bel-7402/ADM cells.Half lethal concentration(IC50)of ADM was determined by MTT assay and apoptosis was detected by AnnexinⅤ-FITC flow cytometry in the Bel-7402/ADM cells.The mRNA and protein levels of HMGB1 expression were determined by qRT-PCR and western blot,respectively.On the other hand,western blot also was applied to assay the protein levels of the receptor for advanced glycation end-products(RAGE),nuclear factor-κB(NF-κB)p-P65 and Bcl-2.The activities of Caspase-9,Caspase-3 were measured with a colorimetric assay kit.Data were analyzed by SPSS 22.0,one-way analysis of variance(ANOVA)and LSD-t test were used to compare continuous variables among groups.Results The levels of protein and mRNA of HMGB1 in the Bel-7402/ADM transfected with HMGB1 siRNA were significantly lower than that in control group and Si-NC group(0.42±0.06 vs 1.00±0.15,1.04±0.22,P=0.004,P=0.003;0.11±0.02 vs 0.33±0.03,0.30±0.02,P<0.001,P<0.001).Compared with the control group and Si-NC group,the expression of RAGE(0.39±0.02 vs 0.51±0.03,P=0.014;0.39±0.02 vs 0.51±0.07,P=0.014),p-P65(0.32±0.04 vs 0.74±0.05,P<0.001;0.32±0.04 vs 0.76±0.06,P<0.001),and Bcl-2(0.50±0.05 vs 1.18±0.09,P<0.001;0.50±0.05 vs 1.06±0.10,P<0.001)were decreased,and the activities of Caspase-9,Caspase-3 were increased significantly in Si-HMGB1 group(3.17±0.38 vs 0.58±0.09,P<0.001;3.17±0.38 vs 0.53±0.11,P<0.001;3.42±0.40 vs 0.68±0.11,P<0.001;3.42±0.40 vs 0.69±0.09,P<0.001).The apoptosis rate of the Bel-7402/ADM cells was increased(17.63±4.52 vs 5.46±0.63,P<0.001;17.63±4.52 vs 5.50±0.61,P<0.001;65.19±5.46 vs 29.73±6.12,P<0.001;65.19±5.46 vs 32.11±4.31,P<0.001),and IC50 of ADM to Bel-7402/ADM decreased from 4.05μg/ml to 1.04μg/ml after transfection of HMGB1 SiRNA.Conclusions HMGB1 suppressed by siRNA can enhance the sensitivity of cells BEL-7402/ADM to ADM and reduce ADM resistance in the cells.The mechanism may be related to the upregulation of adriamycin-induced apoptosis mediated by NF-κB signal pathway in Bel-7402/ADM cells.