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

发布时间：2018-01-17 17:12

本文关键词：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　出处：《临床肝胆病杂志》2016年12期 　论文类型：期刊论文

【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(P0.01)。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,P0.001);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,P0.001);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。
[Abstract]:Objective to investigate the intervention of phosphatidylinositol proteoglycan (GPC). 3 the inhibitory effect of gene transcription and antitumor drugs on the proliferation of hepatoma cells. Methods four kinds of GPC-3-shRNA plasmids were constructed and transfected into HCC HepG2 cells. The expression levels of GPC-3 gene and protein were detected by realtime-PCR Western Blot assay, and the relationship between the expression of GPC-3 gene and the proliferation of hepatoma cells was analyzed. The correlation of apoptosis. The comparison between the two groups was carried out by independent sample t-test. Results among the four transfection plasmids, the efficiency of shRNA1 transfection into HepG2 cells was 85%, and the silencing efficiency at mRNA level was 89.3%. The expression of GPC-3 protein significantly inhibited the inhibition rate of HepG2 cells in the interference group of P0.01and shRNA1 for 72 h, and the inhibition rate was 71.1%. Compared with the negative group, the difference was statistically significant (P 0.001). The biological characteristics of hepatoma cells in shRNA1 interference group were changed, the inhibition rate of migration was 89.1, which was significantly slower than that in negative group, and the difference was statistically significant (P 0.001). The inhibition rate of HepG2 cell motion and invasion was 53.6% and 60.1% respectively. The difference was statistically significant compared with the negative group (52.400% 48.245). The inhibition rate of 尾 -catenin mRNA was 46.9% and the up-regulation rate of Gli1 mRNA was 7.4%. Compared with the control group, the difference was statistically significant (t = 30.108) -3.551 ~ (-1) P = 0.001 ~ (0.009), respectively, at 24 h. The inhibitory rate of 10 渭 mol/L sorafenib combined with shRNA1 was 52.6 渭 mol/L and 100 渭 mol/L with shRNA1. The inhibition rate of hepatoma cells was 79.5%, and the difference was statistically significant compared with the control group (23.314% 50.352). The median inhibitory concentration of Solafenib on HepG2 cells was 4.67 卤1.20 渭 mol/L. Rapamycin was 7.85 卤2.00 nmol / L, and erlotinib was 18.36 卤0.56 渭 mol/L. The inhibition rate of HepG2 cells in combination with shRNA1 was 95.110.Conclusion specific shRNA intervention in GPC-3 transcription can inhibit the proliferation, migration and invasion of hepatoma cells. It also induced apoptosis of hepatoma cells and inhibited the proliferation of cancer cells in cooperation with antitumor drugs, suggesting that GPC-3 may be an effective target for the treatment of HCC, and the combination of targeted therapy will provide a better treatment strategy for HCC.
【作者单位】：南通大学药学院;南通大学医学院;南通大学附属医院;
【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)
【分类号】：R735.7
【正文快照】： 肝细胞癌(HCC)是我国长江口地区常见的恶性肿瘤之一[1-2]。近年来发现,成人正常肝组织中磷脂酰肌醇蛋白聚糖(glypican,GPC)3未见明显表达,胎肝和肝癌组织则高表达。GPC-3通过糖基磷脂酰肌醇锚定于细胞膜上[3-4],且位于Wnt/β-catenin和Hedgehog(Hh)信号通路上游[5-6],导致Wnt/

【参考文献】