干预磷脂酰肌醇蛋白多糖3基因转录联合抗肿瘤药物抑制肝癌细胞增殖的协同作用  

Synergistic effect of intervention of glypican-3 gene transcription combined with antitumor drugs in inhibiting hepatoma cell proliferation

作　　者：杨婕[1] 姚敏[2] 王理[2] 董志珍[3] 顾娟娟[2] 邱历伟[3] 张伟[1] 姚登福[3] 

机构地区：[1]南通大学药学院,江苏南通226001 [2]南通大学医学院,江苏南通226001 [3]南通大学附属医院,江苏南通226001

出　　处：《临床肝胆病杂志》2016年第12期2347-2352,共6页Journal of Clinical Hepatology

基　　金：国家自然科学基金(81673241);江苏省"六大人才高峰"项目(2014-YY-028)

摘　　要：目的探讨干预磷脂酰肌醇蛋白多糖(GPC)3基因转录和联合抗肿瘤药物对肝癌细胞增殖的抑制效果。方法构建4种GPC-3-shRNA质粒转染肝癌HepG2细胞,以realtime-PCR、Western Blot法观察GPC-3基因及蛋白表达水平,分析其与肝癌细胞增殖、凋亡的关系。计量资料两组间比较采用独立样本t检验,多组间比较采用单因素方差分析。结果 4种转染质粒中shRNA1转染HepG2细胞效率>85%,在mRNA水平的沉默效率为89.3%,GPC-3蛋白表达显著抑制(P<0.01)。shRNA1干扰组72 h HepG2细胞抑制率71.1%,与阴性组比较差异有统计学意义(t=18.092,P<0.001);shRNA1干扰组肝癌细胞发生生物学特性改变,迁移抑制率为89.1%,明显慢于阴性组,差异有统计学意义(t=8.326,P<0.001);HepG2细胞运动抑制率为53.6%、侵袭抑制率60.1%,与阴性组比较差异均有统计学意义(t值分别为52.400、48.245,P值均<0.001)。shRNA1干扰组β-catenin mRNA抑制率为46.9%,Gli1 mRNA上调率为7.4%,与对照组比较差异均有统计学意义(t值分别为30.108、-3.551,P值分别为<0.001、0.009)。在24 h时,10μmol/L索拉非尼与shRNA1联合,对肝癌细胞抑制率为52.6%,100μmol/L索拉非尼与shRNA1联合,对肝癌细胞的抑制率为79.5%,与对照组比较差异有统计学意义(t值分别为23.314、50.352,P值均<0.001)。索拉非尼对HepG2细胞的半数抑制浓度(IC_(50))值为(4.67±1.20)μmol/L、雷帕霉素为(7.85±2.00)nmol/L、厄洛替尼为(18.36±0.56)μmol/L,与shRNA1联合使用后HepG2细胞抑制率达95.11%。结论特异性shRNA干预GPC-3转录可抑制肝癌细胞增殖、迁移运动和侵袭能力,并诱导肝癌细胞凋亡,与抗肿瘤药物协同抑制癌细胞增殖,提示GPC-3可能是肝癌治疗有效靶点,联合靶向治疗将为肝癌提供更佳治疗策略。Objective To investigate the inhibitory effect of intervention of glypican- 3（ GPC3） gene transcription combined with antitumor drugs on hepatoma cell proliferation. Methods Four types of GPC3- shRNA plasmids were established and transfected into HepG2 hepatoma cells. Quantitative real- time PCR and Western blot were used to measure the mRNA and protein expression of GPC3 to analyze its association with hepatoma cell proliferation and apoptosis. The independent samples t- test was used for comparison of continuous data between any two groups,and a one- way analysis of variance was used for comparison between multiple groups. Results Among these four plasmids,shRNA1 had a transfection efficiency of 85% in the transfection of HepG2 cells and a silence efficiency of 89. 3% at the mRNA level,and the protein expression of GPC3 was significantly inhibited（ P〈0. 01）. At 72 hours,the GPC3- shRNA1 co- intervention group had an HepG2 cell inhibition rate of 71. 1%,significantly different from that in the negative group（ t = 18. 092,P〈0. 001）,an inhibition rate of migration of 89. 1%,significantly lower than that in the negative group（ t = 8. 326,P〈0. 001）,and inhibition rates of HepG2 cell movement and invasion of 53. 6% and 60. 1%,which were significantly different from those in the negative group（ t = 52. 400 and 48. 245,both P〈0. 001）. The GPC3- shRNA1 co- intervention group had a β- catenin mRNA inhibition rate of 46. 9% and a Gli1 mRNA upregulation rate of 7. 4%,significantly different from those in the negative group（ t = 30. 108 and- 3. 551,P〈0. 001 and P = 0. 009）. At 24 hours,10 μmol/L sorafenib combined with shRNA1 had an inhibition rate of tumor cells of 52. 6% and 100 μmol/L sorafenib combined with shRNA1 had an inhibition rate of tumor cells of 79. 5%,which were significantly different from that in the control group（ t = 23. 314 and50. 352,both P〈0. 001）. The half- maximal inhibitory concentrations of sorafenib,rapamycin,and erlotinib for HepG2 were 4. 67 ± 1. 20μmo

关 键 词：肝肿瘤 磷脂酰肌醇蛋白聚糖类 抗肿瘤药 基因 抑制 药物协同作用 

分 类 号：R735.7[医药卫生—肿瘤]