FOXP3调控非小细胞肺癌A549细胞对阿霉素敏感性的作用及其机制

Regulatory effect of FOXP3 on chemosensitivity of non small-cell lung cancer A549 cells to doxorubicin and its mechnism

目的:探讨叉头蛋白3(FOXP3)基因沉默后非小细胞肺癌(NSCLC)A549细胞对阿霉素(Dox)敏感性的变化,阐明其参与Dox耐药的机制。方法:采用脂质体法将FOXP3小干扰RNA(siRNA)片段转染至人NSCLC A549细胞,细胞分为空白对照组(不转染)、si-NC组(转染对照siRNA)和si-FOXP3组(转染FOXP3-siRNA)。采用Westernblotting法和免疫荧光法检测各组A549细胞中FOXP3蛋白表达水平,CCK-8法检测各组A549细胞增殖活性和半数抑制浓度(IC50)值。各组A549细胞中分别加入0、10和20μmol·L^(-1)DAPT,作为0、10和20μmol·L^(-1)DAPT组;另取A549细胞,分别加入0μmol·L^(-1)DAPT、1.0mg·L^(-1)Dox和1.0mg·L^(-1)Dox联合10μmol·L^(-1)DAPT,作为0μmol·L^(-1)DAPT组、1.0 mg·L^(-1)Dox组和1.0 mg·L^(-1)Dox联合10μmol·L^(-1)DAPT组。Western blotting法检测各组细胞中Notch1、Hes1和FOXP3蛋白表达水平,CCK-8和Western blotting法检测0、10和20μmol·L^(-1)DAPT组A549细胞的IC50值和细胞中Notch1、Hes1及FOXP3蛋白表达水平,Western blotting法检测0μmol·L^(-1)DAPT组、1.0 mg·L^(-1)Dox组和1.0 mg·L^(-1)Dox联合10μmol·L^(-1)DAPT组A549细胞中FOXP3、P-糖蛋白(P-gp)、Notch1和Hes1蛋白表达水平。结果:与空白对照和si-NC组比较,si-FOXP3组A549细胞中FOXP3蛋白表达水平明显降低(P<0.01),增殖活性和IC50值降低(P<0.05或P<0.01),细胞中Notch1、Hes1和FOXP3蛋白表达水平明显降低(P<0.01)。与0μmol·L^(-1)DAPT组比较,10和20μmol·L^(-1)DAPT组A549细胞中Notch1、Hes1和FOXP3蛋白表达水平明显降低(P<0.01)。与0μmol·L^(-1)DAPT组比较,1.0 mg·L^(-1)Dox组A549细胞中FOXP3、P-gp、Notch1和Hes1蛋白表达水平明显升高(P<0.01);与1.0 mg·L^(-1)Dox组比较,1.0 mg·L^(-1)Dox联合10μmol·L^(-1)DAPT组A549细胞中FOXP3、P-gp、Notch1和Hes1蛋白表达水平明显降低(P<0.01)。结论:沉默FOXP3可提高NSCLC细胞对Dox的敏感性,其作用机制与抑制Notch1/Hes1信号通路有关。

Objective:To discuss the change of sensitivity of the non-small-cell lung cancer(NSCLC)A549 cells to doxorubicin(Dox)after silencing forkhead protein 3(FOXP3)gene,and to clarify its mechanism involved in Dox resistance.Methods:The human NSCLC A549 cells were transfected with FOXP3 small interfering RNA(siRNA)by lipofectamine method.The cells were divided into blank control group(without transfection),si-NC group(transfected with control-siRNA),and si-FOXP3 group(transfected with FOXP3-siRNA).Western blotting and immunofluorescence methods were used to detect the expression levels of FOXP3 protein in the A549 cells in various groups;the proliferation activities and half-maximal inhibitory concentration(IC50)values of the A549 cells in various groups were detected by CCK-8 method.The A549 cells were treated with 0,10,and 20μmol·L^(-1) DAPT,and regarded as 0,10,and 20μmol·L^(-1) DAPT groups,respectively.Additionally,the A549 cells were treated with 0μmol·L^(-1) DAPT,1.0 mg·L^(-1) Dox,and 1.0 mg·L^(-1) Dox combined with 10μmol·L^(-1) DAPT,and regarded as 0μmol·L^(-1) DAPT,1.0 mg·L^(-1) Dox,and 1.0 mg·L^(-1) Dox combined with 10μmol·L^(-1) DAPT groups,respectively.Western blotting method was used to detect the expression levels of Notch1,Hes1,and FOXP3 proteins in the A549 cells in various groups;the IC50 values and expression levels of Notch1,Hes1,and FOXP3 proteins in the A549 cells in 0,10,and 20μmol·L^(-1) DAPT groups were detected by CCK-8 and Western blotting methods;the expression levels of FOXP3,P-glycoprotein(P-gp),Notch1,and Hes1 proteins in the A549 cells in 0μmol·L^(-1) DAPT,1.0 mg·L^(-1) Dox,and 1.0 mg·L^(-1) Dox combined with 10μmol·L^(-1) DAPT groups were detected by Western blotting method.Results:Compared with blank control and si-NC groups,the expression level of FOXP3 protein in the A549 cells in si-FOXP3 group was significantly decreased(P<0.01),the proliferative activity and IC50 value were decreased(P<0.05 or P<0.01),and the expression levels of Notch1,Hes1 and FOXP3 proteins were significantly decreased(P<0.01).Compared with 0μmol·L^(-1) DAPT group,the expression levels of Notch1,Hes1,and FOXP3 proteins in the A549 cells in 10 and 20μmol·L^(-1) DAPT groups were significantly decreased(P<0.01).Compared with 0μmol·L^(-1) DAPT group,the expression levels of FOXP3,P-gp,Notch1,and Hes1 proteins in the A549 cells in 1.0 mg·L^(-1) Dox group were significantly increased(P<0.01).Compared with 1.0 mg·L^(-1) Dox group,the expression levels of FOXP3,P-gp,Notch1,and Hes1 proteins in the A549 cells in 1.0 mg·L^(-1) Dox combined with 10μmol·L^(-1) DAPT group were significantly decreased(P<0.01).Conclusion:Silencing FOXP3 can enhance the sensitivity of the NSCLC cells to Dox,and its mechanism is related to the inhibition of the Notch1/Hes1 signaling pathway.