干预NF-κB转录联合二甲双胍对肝癌细胞多药耐药逆转的影响  

Effect of collaboration of interfering NF-κB gene transcription and metformin on reversing multidrug resistance of hepatocellular carcinoma

作　　者：时运[1] 蔡胤[1] 杨绪莉 顾娟娟[1] 杨君伶[1] 吴玮[1] 姚登福[1] 

机构地区：[1]南通大学附属医院临床医学研究中心,南通226001

出　　处：《南通大学学报（医学版）》2015年第4期260-264,共5页Journal of Nantong University(Medical sciences)

基　　金：国家国际科技合作专项(2013DFA32150);江苏省临床医学科技专项(BL2012053);江苏省"六大人才"项目(2013-WSN-078);南通市科技项目(HS2013007)

摘　　要：目的:干预核因子-κB(nuclear factor-kappa B,NF-κB)活化并联合二甲双胍,观察对肝细胞性肝癌(hepatocellular carcinoma,HCC)细胞多药耐药(multidrug resistance,MDR)逆转的影响与机制。方法 :构建与筛选有效质粒p CMV-NF-κB-mi RNA转染和(或)以二甲双胍处理肝癌Hep G2和Hep G2/ADM细胞,采用Western Blot和荧光定量PCR检测蛋白和基因的表达,以CCK-8法分析细胞增殖,以Annexin-Ⅴ-PE/7-ADD双染法检测细胞凋亡。结果:肝癌Hep G2和Hep G2/ADM细胞P-糖蛋白(P-glycoprotein,P-gp)过表达与mdr1 m RNA(3.310±0.154)和NF-κB m RNA(2.580±0.040)表达密切相关;特异性mi RNA干扰NF-κB基因转录并显著下调P-gp的表达,使肝癌细胞对阿霉素敏感增加;二甲双胍预处理Hep G2/ADM细胞后对阿霉素敏感证实通过NF-κB下调P-gp机制;mi RNA干扰与二甲双胍同时应用显著下调NF-κB转录和P-gp的表达,逆转肝癌MDR具有协同作用,且抑制细胞增殖,并促进细胞凋亡。结论:以mi RNA和二甲双胍经NF-κB通路下调MDR1/P-gp表达,有助于肝癌MDR的逆转。Objective: To interfere the activation of nuclear factor-κB(nuclear factor- kappa B, NF-κB) plus metformin to investigate the effect of reversing multidrug resistance(MDR) in hepatocellular carcinoma(HCC) and its mechanism. Methods:The expressions of P-glycoprotein(P-gp) and NF-κB in Hep G2 and Hep G2/ADM cells were inhibited by the most effective plasmid p CMV-NF-κB-mi RNA transfection combining metformin and quantified by Western Blot at protein level or fluorescence quantitative PCR(q RT-PCR) at RNA level. Cell viability was tested by CCK-8 assay. Cell cycle or cell apoptosis was measured by flow cytometry or Annexin- Ⅴ-PE/7-ADD double staining assay. Results: The P-gp overexpressions in Hep G2 and Hep G2/ADM cells were closely related to mdr1 m RNA(3.310 ±0.154) and NF-κB m RNA(2.580 ±0.040). The activity of NF-κB gene transcription was inhibited by specific mi RNA with significantly down- regulation of P-gp expression and enhancing the chemo-sensitivity of HCC cells to adriacin. The Hep G2/ADM cells after pretreatment with meformin were sensitized to adriacin and confirmed with the mechanism of P-gp downregulation through the NF-κB signal pathway. The synergistic effect of both collaboration were founded in Hep G2/ADM cells with cell proliferation inhibition, inducing cell apoptosis and the same molecular mechanism that inhibited the P-gp expression via NF-κB signaling pathway. Conclusion:The present data suggested that mi RNA and meformin could effectively reverse MDR in HCC by downregulation of MDR1/Pgp expression through the NF-κB signaling pathway.

关 键 词：肝细胞性肝癌 多药耐药 逆转 P-糖蛋白 核因子-ΚB 二甲双胍 

分 类 号：R735.7[医药卫生—肿瘤]