西格列汀调节FOXO3-FOXM1信号通路对肺癌细胞化疗敏感性的影响及机制研究

Study on the Effect and Mechanism of Sitagliptin on Chemotherapy Sensitivity of Lung Cancer Cells by Regulating the FOXO3-FOXM1 Signal Pathway

目的:研究西格列汀(SIT)调节叉头框蛋白O3(FOXO3)-叉头蛋白M1(FOXM1)信号通路对肺癌细胞化疗敏感性的影响及机制。方法:体外培养人肺癌细胞A549及其顺铂(DDP)耐药细胞A549/DDP,均以0、0.5、1、2、3、4mmoL/L的SIT处理,以CCK-8法测定各组A549及A549/DDP细胞活力并筛选出SIT最佳作用浓度。将A549/DDP细胞随机分为对照组、SIT(2mmoL/L)组、si-NC组(转染空载质粒)、SIT(2mmoL/L)+si-FOXO3(转染FOXO3 siRNA质粒)组,以SIT和质粒分组处理的同时以0、2、4、8、16、32mg/L DDP处理,然后以CCK-8法测定各组细胞化疗耐药指数。将A549/DDP细胞随机分为对照组、SIT(2mmoL/L)组、DDP(4mg/L)组、DDP(4mg/L)+si-NC组、DDP(4mg/L)+SIT(2mmoL/L)组、DDP(4mg/L)+SIT(2mmoL/L)+si-FOXO3组,分组处理后分别以CCK-8法、克隆形成实验及流式细胞实验测定各组A549/DDP细胞增殖、凋亡;以免疫印迹法检测各组A549/DDP细胞增殖(C-myc、PCNA)、凋亡{Bax、cleaved多聚ADP核糖聚合酶(PARP)}、耐药{P-糖蛋白(P-gp)、多药耐药相关蛋白(MRP1)、MRP2、乳腺癌耐药蛋白(BCRP)}与用FOXO3-FOXM1通路相关蛋白表达。结果:与对照组相比,SIT组细胞化疗耐药指数降低(P<0.05),si-NC组细胞化疗耐药指数无明显变化(P>0.05);与SIT组相比,SIT+si-FOXO3组细胞化疗耐药指数升高(P<0.05)。与对照组相比,DDP组、DDP+si-NC组A549/DDP细胞活力、克隆形成率、C-myc及PCNA蛋白表达降低(P<0.05),凋亡率、Bax及cleaved PARP蛋白表达升高(P<0.05);DDP+SIT组A549/DDP细胞活力、克隆形成率、C-myc及PCNA、P-gp、MRP1、MRP2、BCRP、FOXM1蛋白表达降低(P<0.05),凋亡率、Bax及cleaved PARP、FOXO3蛋白表达升高(P<0.05);SIT组A549/DDP细胞P-gp、MRP1、MRP2、BCRP、FOXM1蛋白表达降低(P<0.05),FOXO3蛋白表达升高(P<0.05)。与DDP组相比,DDP+SIT组A549/DDP细胞活力、克隆形成率、C-myc及PCNA、P-gp、MRP1、MRP2、BCRP、FOXM1蛋白表达降低(P<0.05),凋亡率、Bax及cleaved PARP、FOXO3蛋白表达升高(P<0.05);DDP+si-NC组A549/DDP细胞各指标无明显变化(P>0.05)。与DDP+SIT组相比,DDP+SIT+si-FOXO3组A549/DDP细胞活力、克隆形成率、C-myc及PCNA、P-gp、MRP1、MRP2、BCRP、FOXM1蛋白表达升高(P<0.05),凋亡率、Bax及cleaved PARP、FOXO3蛋白表达降低(P<0.05)。结论:SIT可通过促进FOXO3-FOXM1信号传导而下调耐药蛋白表达,增强肺癌细胞化疗敏感性,进而提高DDP对肺癌DDP耐药细胞的杀伤作用。

Objective:To investigate the effect and mechanism of sitagliptin(SIT)on the chemosensi-tivity of lung cancer cells by regulating the forkhead box protein O3(FOXO3)-forkhead protein M1(FOXM1)signaling pathway.Methods:Human lung cancer cell line A549 and cisplatin(DDP)resistant cell line A549/DDP were cultured in vitro and treated with 0,0.5,1,2,3,and 4 mmol/L of SIT.CCK-8 method was applied to determine the A549 and of A549/DDP cells viability in each group to screen the opti-mal concentration of SIT.A549/DDP cells were randomly separated into control group,SIT(2mmoL/L)group,an si-NC group(transfected with si-NC plasmid),a SIT(2mmol/L)+si-FOXO3(transfected with FOXO3 siRNA plasmid)group,and were treated with 0,2,4,8,16,and 32 mg/L DDP while treating with SIT and plasmid,then the CCK-8 method was applied to determine the chemotherapy resistance index of cells in each group.A549/DDP cells were randomly separated into control group,SIT(2mmol/L)group,DDP(4mg/L)group,DDP(4mg/L)+si-NC group,DDP(4mg/L)+SIT(2mmol/L)group,and a DDP(4mg/L)+SIT(2mmol/L)+si-FOXO3 group,after grouping,the CCK-8 method,clone formation assay,and flow cytometry assay were applied to determine the proliferation and apoptosis of A549/DDP cells in each group;Western blot was applied to detect the expression of A549/DDP cell proliferation(C-myc,PCNA),apoptosis{Bax,cleaved poly ADP ribose polymerase(PARP)},drug resistance{P-glycoprotein(P-gp),multidrug resistance associated protein(MRP1),MRP2,breast cancer drug resistance protein(BCRP)}and FOXO3-FOXM1 pathway related proteins in each group.Results:Compared with the control group,the chemotherapy resistance index of cells in the SIT group decreased(P<0.05),while there was no significant change in the chemotherapy resistance index of cells in the si-NC group(P>0.05);compared with the SIT group,the SIT+si-FOXO3 group showed an increase in cell chemotherapy resistance index(P<0.05).Compared with the control group,the A549/DDP cell viability,clone formation rate,C-myc,and PCNA protein expression in the DDP group and DDP+si-NC group reduced(P<0.05),the apoptosis rate,Bax and cleaved PARP pro-tein expression was up-regulated(P<0.05);the A549/DDP cell viability,clone formation rate,C-myc and PCNA,P-gp,MRP1,MRP2,BCRP,and FOXM1 protein expression in the DDP+SIT group reduced(P<0.05),the apoptosis rate,Bax and cleaved PARP,FOXO3 protein expression was up-regulated(P<0.05);the P-gp,MRP1,MRP2,BCRP,and FOXM1 protein expression in A549/DDP cells in the SIT group de-creased(P<0.05),the FOXO3 protein expression was up-regulated(P<0.05).Compared with the DDP group,the A549/DDP cell viability,clone formation rate,C-myc and PCNA,P-gp,MRP1,MRP2,BCRP,and FOXM1 protein expression in the DDP+SIT group reduced(P<0.05),the apoptosis rate,Bax and cleaved PARP,FOXO3 protein expression was up-regulated(P<0.05);there was no significant change in all indicators of A549/DDP cells in the DDP+si-NC group(P>0.05).Compared with the DDP+SIT group,the A549/DDP cell viability,clone formation rate,C-myc and PCNA,P-gp,MRP1,MRP2,BCRP,and FOXM1 protein expression in the DDP+SIT+si-FOXO3 group increased(P<0.05),the apopto-sis rate,Bax and cleaved PARP,FOXO3 protein expression decreased(P<0.05).Conclusion:SIT can down-regulate the expression of drug resistant proteins,and enhance the chemotherapy sensitivity of lung cancer cells by promoting FOXO3-FOXM1 signaling,and thereby enhance the killing effect of DDP on DDP resistant lung cancer cells.