干预胰岛素样生长因子Ⅰ型受体活化联合抗癌药物抑制肝癌细胞增殖的协同作用分析

Synergistic effect of interventing insulin-like growth factor-Ⅰ receptor activation combined with anti-cancer drugs in inhibiting the proliferation of hepatocellular carcinoma cells

目的探讨干预胰岛素样生长因子Ⅰ型受体(IGF-ⅠR)基因转录,对抑制肝癌细胞增殖药物的协同作用。方法以p GPU6/GFP/Neo-IGF-ⅠR-shRNA高效质粒,转染IGF-ⅠR高表达的HBV阳性肝癌细胞PLC/PRF/5和HBV阴性Bel-7404,设空白对照组、阴性对照组和IGF-ⅠR-shRNA干预组;以荧光定量逆转录PCR和Western Blot分析RNA和蛋白表达;以细胞计数试剂盒分析细胞增殖;以流式细胞术、Annexin-V-PE/7-ADD分析细胞周期与凋亡。计量资料组间比较采用t检验,计数资料组间比较采用Fisher确切概率法。结果 IGF-ⅠR shRNA转染肝癌细胞PLC/PRF/5效率为71%、肝癌细胞Bel-7404为90%,2种肝癌细胞IGF-ⅠR在mRNA和蛋白表达水平上同步减少;干预组细胞较阴性对照组均明显抑制,2组间肝癌细胞Bel-7404 72h时抑制率比较差异有统计学意义[(61.5±1.7)%vs(11.2±0.9)%,t=5.493,P<0.05],2组间肝癌细胞PLC/PRF/5 72 h时抑制率比较差异有统计学意义[(63.9±3.9)%vs(9.5±1.1)%,t=19.244,P<0.001]。干预组肝癌细胞Bel-7404凋亡率显著高于空白对照组(35.96%vs 12.16%,P<0.001)和阴性对照组(35.96%vs 9.43%,P<0.001),干预组肝癌细胞PLC/PRF/5凋亡率显著高于空白对照组(44.84%vs 6.62%,P<0.001)和阴性对照组(44.84%vs 4.02%,P<0.001);干预组肝癌细胞Bel-7404和PLC/PRF/5 G0/G1期百分率均明显高于阴性对照组[(59.0±1.3)%vs(48.4±0.8)%,t=12.032,P<0.001;(65.4±0.5)%vs(53.5±0.7)%,t=22.789,P<0.001)];干预组肝癌细胞Bel-7404和PLC/PRF/5周期蛋白cyclin D1表达均明显低于阴性对照组[(59.6±4.7)%vs(90.0±3.4)%,t=7.389,P<0.05;(39.9±0.5)%vs(90.2±14.6)%,t=4.876,P<0.05)];肝癌细胞Bel-7404和PLC/PRF/5干预组与阴性对照组在索拉非尼浓度分别为0、2.5、5、10、20 nmol/L和奥沙利铂浓度分别为0、5、10、20和40μmol/L时的光密度值比较差异均有统计学意义(P值均<0.05)。结论下调IGF-ⅠR基因转录,具有抑制肝癌细胞增殖、改善药物敏感性的协同作用。

Objective To investigate the intervention of gene transcription of insulin- like growth factor- Ⅰ receptor（ IGF- ⅠR） and its synergistic effect with anti- cancer drugs in inhibiting the proliferation of hepatocellular carcinoma（ HCC） cells. Methods The HBV-positive HCC PLC / PRF /5 and HBV- negative Bel- 7404 cells were transfected with the efficient plasmid p GPU6 / GFP / Neo- IGF- ⅠR-shRNA. Fluorescent quantitative RT- PCR and Western blot were used to measure mRNA and protein expression,the Cell Counting Kit-8 was used to analyze cell proliferation,and flow cytometry and Annexin- V- PE /7- ADD were used to analyze cell cycle and apoptosis.The t- test was used for comparison of continuous data between groups,the Fisher＇s exact test was used for comparison of categorical data between groups. Results The efficiency of IGF-ⅠR shRNA transfection was 71% in HCC PLC / PRF /5 cells and 90% in Bel- 7404 cells,and both cells showed reductions in the mRNA and protein expression of IGF- ⅠR. The intervention group showed a significant inhibition compared with the negative control group,and the 72- hour inhibition rates of Bel- 7404 cells and PLC / PRF /5 cells showed significant differences between the two groups（ inhibition rates of Bel- 7404 cells： 61. 5% ± 1. 7% vs 11. 2% ± 0. 9%,t = 5. 493,P〈0. 05; inhibition rates of PLC / PRF /5 cells： 63. 9% ± 3. 9% vs 9. 5% ± 1. 1%,t = 19. 244,P〈0. 001）. The intervention group showed a significantly higher apoptosis rate of Bel- 7404 cells than the blank control group（ 35. 96% vs 12. 16%,P〈0. 001） and the negative control group（ 35. 96% vs 9. 43%,P〈0. 001）,as well as a significantly higher apoptosis rate of PLC/PRF/5 cells than the blank control group（ 44. 84% vs 6. 62%,P〈0. 001） and the negative control group（ 44. 84% vs 4. 02%,P〈0. 001）. The co- intervention group showed significantly higher percentages of Bel- 7404 cells and PLC / PRF /5 cells in G0/ G1 phase than the negative control group（ 59. 0% ± 1. 3%vs 48. 4% ± 0. 8%,t = 12. 032,P〈0. 001; 65. 4% ± 0. 5% vs 53. 5% ± 0. 7%,t = 22. 789,P〈0. 001）. The co- intervention group showed significantly lower expression of cyclin D1 in Bel- 7404 cells and PLC / PRF /5 cells than the negative control group（ 59. 6% ± 4. 7%vs 90. 0% ± 3. 4%,t = 7. 389,P〈0. 05; 39. 9% ± 0. 5% vs 90. 2% ± 14. 6%,t = 4. 876,P〈0. 05）. The OD value of Bel- 7404 cells and PLC / PRF /5 cells showed a significant difference between the intervention group and the negative control group when the sorafenib concentration was 0,2. 5,5,10,and 20 nmol / L and the oxaliplatin concentration was 0,5,10,20,and 40 μmol / L（ all P〈0. 05）. Conclusion The downregulation of IGF-ⅠR gene transcription has the synergistic effect of inhibiting HCC cell proliferation and improving drug susceptibility.