慢病毒介导靶向P27RF-Rho基因沉默对肝癌细胞侵袭性的影响

摘要

目的：探讨慢病毒靶向介导技术沉默 P27RF-Rho 基因，阐述其对肝癌细胞侵袭性的影响。方法：构建P27RF-Rho RNAi慢病毒。慢病毒感染肝癌细胞 BEL7402。实验分为 P27RF-Rho-siRNA 实验组、Scramble-siRNA阴性对照组和BEL7402空白对照组。Western bloting法检测P27RF-Rho基因沉默效果及肝癌相关蛋白 RhoA、RhoC、VEGF、P53和PTEN表达水平；明胶酶谱分析肿瘤侵袭相关基质金属蛋白酶（MMPs）活性；细胞划痕和体外Transwell小室侵袭实验比对细胞迁移和侵袭能力的改变。结果：Western blotting法检测，P27RF-Rho-siRNA实验组BEL7402中P27RF-Rho、RhoA、RhoC和 VEGF蛋白表达水平明显低于2个对照组（P＜0.05），P53和 PTEN表达水平高于2个对照组（P＜0.05）。明胶酶谱，P27RF-Rho-siRNA实验组MMPs活性明显降低（P＜0.01）。划痕实验，P27RF-Rho-siRNA 实验组细胞迁移距离明显低于2个对照组（P＜0.01）。实验组穿过Transwell小室的平均细胞数明显少于2个对照组（P＜0.01）。结论：沉默 P27RF-Rho基因可以降低肝癌细胞BEL7402的侵袭和迁移能力。%Objective:To investigate the silencing of P27RF-Rho gene with lenvirus targeting mediated technique,and to clarify its influence in the invasion of liver cancer cells.Methods:The P27RF-Rho RNAi lentivirus was constructed. The liver cancer BEL7402 cells were infected with lentivirus. The experiment was divided into P27RF Rho-siRNA group, scramble-siRNA group and BEL7402 group.The effect of silencing P27RF-Rho gene and the expression levels of hepatocellular carcinoma (HCC)associated proteins RhoA,RhoC, VEGF,P53 and PTEN were detected;the activities of matrix metalloproteinase (MMPs)associated with tumor invasion were analyzed by Gelatin zymography;the variation of transfer ability and invasion abilities were compared by Wound healing assay experiment and Transwell experiment.Results:The Western blotting results showed the expression levels of P27RF-Rho,RhoA,RhoC,and VEGF proteins in the BEL7402 cells in experiment group were significantly lower than those in two control groups (P<0.05),and the expression levels of P53 and PTEN were higher than those in two control groups (P<0.05).The results of Gelatin zymography showed the activities of MMPs in experiment group were significantly lower than those in two control groups (P<0.01 );Wound healing assay showed that the migration ability of the BEL7402 cells in experiment group was significantly inhibited (P<0.01);the number of cells passed through the Transwell Chambers in experiment group was significantly less than those in two control groups (P<0.01).Conclusion:Silenceing P27RF-Rho can weaken the invasion ability and migration ability of human HCC BEL7402 cells.