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

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. 20μmol/L,7. 85 ± 2. 00 nmol/L,and 18. 36 ± 0. 56 μmol/L,respectively,and their combination with shRNA1 had an HepG2 cell inhibition rate of 95. 11%. Conclusion Intervention of GPC3 gene transcription with specific shRNA can inhibit hepatoma cell proliferation,migration and movement,and invasion ability,induce hepatoma cell apoptosis,and inhibit hepatoma cell proliferation when combined with antitumor drugs in a synergistic manner. This suggests that GPC3 may be an effective therapeutic target for liver cancer and that combined targeted therapy can provide better strategies for the treatment of liver cancer.