HIF-2α-VEGF-Notch信号通路在高强度聚焦超声不完全消融后肝癌中的作用

Role of HIF-2α-VEGF-Notch in angiogenesis of residual hepatocellular carcinoma after high intensity focused ultrasound

目的探讨HIF-1α、HIF-2α、VEGF、Notch等基因在高强度聚焦超声不完全消融肝癌血管新生中的表达及其作用。方法实验共分5组,HIFU残留肝癌细胞亚系为实验组,未处理肝癌细胞为对照组,转染HIF-2α基因沉默重组慢病毒残留肝癌细胞为抑制HIF-2α组,转染VEGF基因沉默重组慢病毒残留肝癌细胞为抑制VEGF组,转染空载慢病毒残留肝癌细胞为空载组。免疫荧光检测实验组与对照组的VEGF表达。低氧(1%O_2)处理实验组与对照组细胞0、6、12、24 h,荧光定量PCR和Western blot检测HIF-1α、HIF-2α、VEGF mRNA和蛋白表达水平。荧光定量PCR和Western blot检测各组细胞HIF-1α、HIF-2α、VEGF、Notch、DLL4 mRNA和蛋白表达水平。结果 VEGF蛋白在对照组与实验组细胞中均有表达。HIF-1α在低氧处理12 h达到顶峰,随后下降,而HIF-2α、VEGF随着低氧处理时间增加而呈上升趋势,且实验组HIF-2α、VEGF表达较对照组明显增高(P<0.05)。转染慢病毒后,抑制HIF-2α组HIF-2α、VEGF mRNA和蛋白表达均较实验组显著下调(P<0.05),抑制VEGF组VEGF、Notch、DLL4 mRNA和蛋白表达均较实验组显著下调(P<0.05),实验组HIF-2α、VEGF、Notch、DLL4mRNA和蛋白表达均高于对照组(P<0.05)。结论 HIF-2α-VEGF-Notch信号通路参与调控高强度聚焦超声残余肝癌血管新生。

Objective To determine the expression of hypoxia-inducible factor-1α（HIF-1α） and HIF-2α,vascular endothelium growth factor（VEGF） and Notch in the residual hepatocellular carcinoma（HCC） after high intensity focused ultrasound（HIFU）. Methods The cell subline was established based on HCC SMMC7721 cells survived from HIFU treatment. Immunofluorescence staining was used to measure the expression of VEGF in normal control cells and HIFU treated cells. Real-time PCR and Western blotting were used to measure the expression of HIF-1α,HIF-2α and VEGF at mRNA and protein levels at 0,6,12 and 24 h under hypoxic condition（1% O_2） in both treated and control cells. Then,the treated cells was infected with lentiviral vectors LV-shRNA-VEGF,LV-shRNA-HIF-2α and LV-shRNA-NC respectively,and the mRNA and protein expression of VEGF,HIF-2α,Notch,and DLL4 was quantified by real-time PCR and Western blotting. Results The VEGF protein was mainly expressed in the cytoplasm in the treated and control cells. The expression of HIF-1α in the treated and control cells reached peak in 12 h after hypoxic treatment and then decreased,while the expression of HIF-2α and VEGF was increased with the time elapse（P〈0. 05）. The mRNA and protein expression of HIF-2α and VEGF was significantly lower in HIF-2αmodulated cells than the treated cells（P〈0. 05）,and that of VEGF,Notch and DLL4 was also decreased in the VEGF modulated cells than that in the treated cells（P〈0. 05）. The levels of HIF-2α,VEGF,Notch and DLL4 were significantly higher in the treated cells than control cells（P〈0. 05）. Conclusion HIF-2α-VEGF-Notch signaling pathway is involved in the regulation of angiogenesis in residual hepatocellular carcinoma after HIFU treatment.