ZBP-89通过HIF-1α/Notch1通路对肝癌干细胞干性的调控作用

The regulatory effect of ZBP-89 on the stem cell dryness of hepatoma via HIF-1α/Notch 1 pathway

目的探究ZBP-89(Zinc-binding protein-89)通过HIF-1α/Notch1通路对肝癌干细胞干性的调控作用。方法构建ZBP-89过表达慢病毒载体(Len-ZBP-89)并进行慢病毒包装,微球体培养法富集肝癌干细胞,流式细胞术检测细胞中EpCAM、CD133的表达,CoCl 2(HIF-1α激活剂)处理肝癌干细胞,RT-PCR检测细胞中ZBP-89、 HIF-1α和Notch1mRNA表达,Westernblot检测细胞中ZBP-89、 HIF-1α、 Notch1、 CD44、CD133和EpCAM蛋白表达,Transwell检测细胞迁移和侵袭能力,光学显微镜观察特征性肿瘤球体的形成。结果 Len-ZBP-89慢病毒包装成功,病毒滴度为1.32×10^(9) TU/mL;肝癌细胞PLC/PRF/5中ZBP-89 mRNA和蛋白表达水平下调,HIF-1α和Notch1 mRNA和蛋白表达水平上调;PLC/PRF/5细胞成功富集肝癌干细胞PLC/PRF/5.C,PLC/PRF/5.C中EpCAM、CD133的表达水平均上调;Len-ZBP-89可下调PLC/PRF/5.C细胞中HIF-1α、Notch1、CD133、CD44和EpCAM的蛋白表达,下调细胞迁移、侵袭能力和特征性肿瘤球体克隆数量;CoCl 2(HIF-1α激活剂)可上调PLC/PRF/5.C细胞中HIF-1α、Notch1、CD133、CD44和EpCAM的蛋白表达,上调细胞迁移、侵袭能力和特征性肿瘤球体克隆数量(P<0.05);CoCl 2处理可逆转Len-ZBP-89对PLC/PRF/5.C干性的抑制作用。结论过表达ZBP-89通过抑制HIF-1α/Notch1通路抑制肝癌干细胞干性。

Objective To investigate the regulatory effect of ZBP-89(Zinc-binding protein-89) on the stemness of hepatocellular carcinoma stem cells via HIF-1α/Notch1 pathway. Methods ZBP-89 overexpression lentiviral vector(Len-ZBP-89) was constructed and packaged. Microsphere culture method was used to enrich liver cancer stem cells. Flow cytometry was used to detect the expression of EpCAM and CD133. Liver cancer stem cells were treated with CoCl2(HIF-1α activator). HIF-1α and Notch1 mRNA expressions were detected by RT-PCR. ZBP-89, HIF-1α, Notch1, CD44, CD133 and EpCAM protein expressions were detected by Western blot. Transwell was used to detect cell migration and invasion ability, and the formation of characteristic tumor spheroids was observed by light microscope. Results The lentiviral vector of ZBP-89 overexpression was successfully constructed, titer of lentivirus after packaging was 1.32×10^(9) TU/mL, ZBP-89 mRNA and protein expression was down-regulated in liver cancer PLC/PRF/5 cells, and HIF-1α and Notch1 mRNA and protein expressions were up-regulated(P<0.05). PLC/PRF/5 cells were successfully enriched for liver cancer stem cells PLC/PRF/5.C, and the expressions of EpCAM and CD133 in PLC/PRF/5.C were all up-regulated;LenZBP-89 down-regulated protein expression levels of HIF-1α, Notch1, CD133, CD44 and EpCAM in PLC/PRF/5.C cells, down-regulated cell migration, invasion ability and number of characteristic tumor spheroid clones;CoCl2(HIF-1α activation agent) up-regulated the protein expression levels of HIF-1α, Notch1, CD133, CD44 and EpCAM in PLC/PRF/5.C cells, up-regulated cell migration, invasion ability and number of characteristic tumor spheroid clones(P<0.05);CoCl2 treatment could reverse the inhibitory effect of Len-ZBP-89 on the stemness of PLC/PRF/5.C. Conclusion Overexpression of ZBP-89 inhibits the stemness of liver cancer stem cells via inhibiting the HIF-1α/Notch1 pathway.