siRNA靶向NRP-1基因沉默经Wnt、ROS及TGF-β1途径抑制宫颈癌细胞增殖

目的：探讨RNA干扰神经纤毛蛋白1（NRP-1）基因对宫颈癌细胞凋亡、ROS水平及免疫分子表达的影响。方法：以人正常宫颈细胞Ect1/E6E7 为对照细胞，Western blot 检测宫颈癌Hela、CaSki、SiHa、HCC94细胞NRP-1的蛋白表达；通过LipofectamineTM2000将NRP-1-siRNA瞬时转染CaSki细胞，并设置阴性对照组（NC-siRNA）和空白对照组（Control组），转染48 h后，CCK8法检测各组细胞活力；流式细胞仪检测各组细胞凋亡率及ROS含量；Western blot检测免疫抑制因子TGF-β1及Wnt信号通路相关蛋白Wnt1、β-catenin、Survivin和Cyclin D1的蛋白表达。结果：宫颈癌细胞中NRP-1的表达均显著高于在Ect1/E6E7细胞中的表达（P<0.05）；转染NRP-1-siRNA的CaSki细胞NRP-1的蛋白表达显著低于对照组（P<0.05）；与对照组比较，NRP-1-siRNA组细胞活力显著降低，细胞凋亡率及ROS含量均显著升高，TGF-β1、Wnt1、β-catenin、Survivin和Cyclin D1的蛋白表达均显著降低（P<0.05）。结论：抑制宫颈癌中NRP-1基因表达可降低癌细胞活力，诱导细胞凋亡，降低免疫抑制分子TGF-β1表达，其机制与提高细胞ROS水平和下调Wnt信号通路有关。

siRNA targeting NRP-1 gene silencing inhibited proliferation of cervical cancer cells through Wnt,ROS and TGF-β1 pathway

Objective:To investigate the effect of RNA interference with neurocilin-1 gene expression on apoptosis,ROS level and expression of immune molecules in cervical cancer cells.Methods: Human normal cervical cells Ect1/E6E7 was control cells,the expression of NRP-1 protein in Hela,CaSki,SiHa,HCC94 cervical cancer cells were detected by Western blot;NRP-1-siRNA were transiently transfected into CaSki cells by LipofectamineTM2000,and negative control group (NC-siRNA) and blank control group (Control group)were set,and cells were transfected for 48 h,cell viability was detected by CCK8;cell apoptosis and ROS contentwere detected by flow cytometry in each group;the expression of TGF-β1,Wnt1,β-catenin,Survivin and Cyclin D1 protein were detected by Western blot.Results: The expression of NRP-1 in cervical cancer cells were significantly higher than the expression in Ect1/E6E7 cells (P<0.05);the expression of NRP-1 proteinin CaSki cells after transfected with NRP-1-siRNA was significantly lower than the control group (P<0.05);compared with the control group,cell viability significantly decreased in NRP-1-siRNA group,cell apoptosis rate and ROS content increased significantly,the expression of TGF-β1,Wnt1,β-catenin,Survivin and Cyclin D1 protein decreased significantly (P<0.05).Conclusion: Inhibition of NRP-1 gene expression in cervical cancer can reduce cell viability,induce apoptosis,and reduce the expression of immunosuppressive molecules TGF-β1,which is related to increase cell ROS level and down regulation of Wnt signaling pathway.