沉默NRF2基因表达及鸦胆子苦醇均可抑制人胆管癌RBE细胞增殖

Silencing NRF 2 gene expression and brusatol both inhibit the proliferation of human cholangiocarcinoma RBE cells

目的:探讨核因子E2相关因子2(nuclear factor E2-related factor 2,NRF2)在人胆管癌细胞株RBE细胞增殖中的作用,以及鸦胆子苦醇(brusatol)对RBE细胞增殖的影响。方法:利用UALCAN数据库检索NRF2在正常人胆道组织和胆管癌组织中的表达情况;分别采用实时荧光定量PCR和蛋白质印迹法检测人胆管癌细胞株RBE和正常胆管上皮H69细胞中NRF2 m RNA和蛋白的表达水平。采用脂质体转染法将靶向NRF2基因的si RNA(si NRF2-1和si NRF2-2)转入RBE细胞,分别采用实时荧光定量PCR和蛋白质印迹法检测RBE细胞中NRF2 m RNA和蛋白的表达情况。采用DCFH-DA探针法检测沉默NRF2表达对RBE细胞中活性氧(reactiveoxygenspecies,ROS)水平的影响。CCK-8法和平板克隆形成实验检测沉默NRF2表达对RBE细胞增殖的影响。最后,再用CCK-8法检测鸦胆子苦醇对RBE细胞增殖的影响,实时荧光定量PCR和DC-FHDA探针法分别检测鸦胆子苦醇对RBE细胞中NRF2 m RNA表达水平的影响以及细胞内ROS水平的影响。结果:NRF2在胆管癌组织中的表达水平明显高于正常胆管组织(P<0.05)。人胆管癌细胞株RBE中NRF2 m RNA和蛋白的表达水平均较正常胆管上皮H69细胞明显上调(P<0.001和P<0.01)。si NRF2-1和si NRF2-2均能明显下调RBE细胞中NRF2 m RNA和蛋白的表达水平均(P均<0.01)。沉默NRF2表达能有效提高RBE细胞中的ROS含量(P均<0.05),而抑制RBE细胞的增殖能力(P均<0.01)。鸦胆子苦醇能够有效抑制RBE细胞的增殖能力(P<0.01),下调RBE细胞中NRF2 m RNA的表达水平(P<0.01)以及提高RBE细胞中的ROS含量(P<0.01)。结论:NRF2在胆管癌中表达增加,沉默NRF2基因表达以及鸦胆子苦醇均能有效提高人胆管癌RBE细胞内ROS的含量并抑制胆管癌细胞增殖,因此NRF2有望成为胆管癌治疗的一个重要靶点,而鸦胆子苦醇有望成为临床上治疗胆管癌的一个新药。

Objective:To investigate the effect of nuclear factor E2-related factor 2(NRF2)on the proliferation of human cholangiocarcinoma RBE cells and the effect of brusatol on the proliferation of RBE cells.Methods:The expression level of NRF2 m RNA in bile duct and cholangiocarcinoma was analyzed by using UALCAN database.The expression levels of NRF2 m RNA and protein in normal bile duct epithelial H69 cells and cholangiocarcinoma RBE cells were detected by real-time fluorescent quantitative PCR and Western blotting,respectively.The si RNA targeting NRF 2 gene(si NRF2-1 and si NRF2-2)was transfected into RBE cells by liposome,and the expression levels of NRF2 m RNA and protein were detected by real-time fluorescent quantitative PCR and Western blotting,respectively.The effect of silencing NRF2 gene expression on the level of reactive oxygen species(ROS)in RBE cells was detected by DCFHDA method,and the proliferation of RBE cells was detected by CCK-8 assay and plate clone formation assay.Finally,the effect of brusatol on the proliferation of RBE cells was detected by CCK-8 assay,and the effect of expression levels of NRF2 m RNA and intracellular ROS level in RBE cells were detected by real-time fluorescence quantitative PCR and DC-FHDA probe assay,respectively.Results:The expression of NRF2 in cholangiocarcinoma tissues was higher than that in normal bile duct tissues.Compared with normal bile duct epithelial H69 cells,the expression levels of NRF2 m RNA and protein in human cholangiocarcinoma RBE cells were significantly up-regulated(P<0.001 and P<0.01).Both si NRF2-1 and si NRF2-2 significantly downregulated the expression levels of NRF2 m RNA and protein in RBE cells(both P<0.01).Brusatol could effectively inhibit the proliferation ability of RBE cells(P<0.01),down-regulate the expression level of NRF2 m RNA in RBE cells(P<0.01),and increase the ROS content in RBE cells(P<0.01).C on cl u si on:NRF2 expression was increased in cholangiocarcinoma,the NRF 2 ge ne expression was silenced,and the brusatol effectively increased the level of ROS in human cholangiocarcinoma RBE cells and inhibit the proliferation of cholangiocarcinoma cells,suggesting NRF2 may be an important target in cholangiocarcinoma treatment,and brusatol may be a new drug in clinical treatment of cholangiocarcinoma.