过表达乙酰肝素酶对胆囊癌细胞增殖及PI3K/AKT信号通路的影响

Effect of overexpression of heparanase on proliferation of gallbladder cancer cells and PI3K/AKT signaling pathway

目的探讨过表达乙酰肝素酶(HPSE)对胆囊癌细胞系GBC-SD细胞增殖及磷脂酰肌醇3-激酶/蛋白质丝氨酸苏氨酸激酶(PI3K/AKT)信号通路的影响。方法构建HPSE真核表达载体,然后获得转基因过表达HPSE的胆囊癌细胞株GBD-SD。通过实时荧光定量聚合酶链反应(qRT-PCR)和Western blot分析GBC-SD和GBD-SD中HPSE mRNA和蛋白表达。通过集落形成实验、MTT来评估各组细胞的增殖情况。采用Western blot分析PI3K/AKT信号通路相关蛋白(p-PI3K、PI3K、p-AKT和AKT)的水平。结果成功转染后的GBD-SD中HPSE mRNA水平和蛋白水平升高,差异有统计学意义(P<0.01)。与正常表达HPSE的GBC-SD相比,过表达HPSE的GBD-SD细胞光密度(OD)值和集落形成能力提高,差异有统计学意义(P<0.05)。Western blot检测结果显示,与GBC-SD细胞相比,过表达HPSE的GBD-SD组细胞p-AKT、p-PI3K蛋白表达水平、p-AKT/AKT值和p-PI3K/PI3K值增加,差异有统计学意义(P<0.01),而总AKT和总PI3K蛋白表达水平无明显变化。GBC-SD细胞经PI3K/AKT通路特异性抑制剂LY294002处理2 d后,其增殖能力和p-AKT/AKT值降低,差异有统计学意义(P<0.01)。结论过表达HPSE能够促进GBC-SD细胞的增殖,这一效应可能与PI3K/AKT信号通路有关。

Objective To explore the effect of overexpression of heparanase(HPSE) on the proliferation of gallbladder cancer cell line GBC-SD and the phosphatidylinositol 3-kinase/protein serine-threonine kinase(PI3 K/AKT) signaling pathway. Methods The HPSE eukaryotic expression vector was constructed, and then the gallbladder cancer cell line GBD-SD with transgenic overexpression of HPSE was obtained. The mRNA and protein expression of HPSE in GBC-SD and GBD-SD were analyzed by quantitative real-time polymerase chain reaction(qRT-PCR) and western blot. The proliferation of cells in each group was assessed by colony formation assay and MTT. The protein levels of PI3 K/AKT signaling pathway related proteins(p-PI3 K, PI3 K, p-AKT and AKT) were analyzed by western blot. Results The mRNA and protein levels of HPSE in GBD-SD after successful transfection were significantly increased(P<0.01). Compared to GBC-SD with normal expression of HPSE, the optical density(OD) value and colony formation ability of GBD-SD cells with overexpression of heparanase were significantly increased(P<0.05). The results of western blot showed that compared with GBC-SD cells, the expression levels of p-AKT, p-PI3 K protein, p-AKT/AKT value and p-PI3 K/PI3 K value in GBD-SD cells with overexpression of HPSE were increased significantly(P<0.01), but the expression levels of total AKT and total PI3 K protein did not change obviously. After 2 days of treatment with specific inhibitor LY294002 for PI3 K/AKT pathway,the proliferation ability and p-AKT/AKT value of GBC-SD cells were significantly decreased( P < 0. 01). Conclusion Overexpression of HPSE can promote the proliferation of GBC-SD cells,which may be related to the PI3 K/AKT signaling pathway.