五味子素通过调控FOXC1基因对结直肠癌细胞恶性生物学行为的作用机制

Study on the Mechanism of Schisandrin on the Malignant Biological Behavior of Colorectal Cancer Cells by Regulating FOXC1 Gene

目的探讨五味子素通过调控叉头框转录因子C1(FOXC1)基因对结直肠癌(CRC)细胞恶性生物学行为的作用机制。方法采用不同浓度(0、12.5、25、50μmol/L)五味子素处理HCT116细胞,筛选合适浓度细胞用于后续实验,随机分层法分为对照组(未做任何处理)、FOXC1-NC组(转染FOXC1模拟物阴性对照)、FOXC1组(转染FOXC1)、五味子素组(添加50μmol/L五味子素)、FOXC1+五味子素组(转染FOXC1,并添加50μmol/L五味子素),流式细胞仪检测HCT116细胞凋亡情况,Transwell实验检测细胞侵袭能力,Realtime-PCR检测FOXC1、上皮型钙黏附素(E-cadherin)、基质金属蛋白酶2(MMP2)、波形蛋白(Vimentin)mRNA表达,Western blot检测FOXC1、MMP2、E-cadherin、Vimentin蛋白表达水平。结果与0μmol/L比较,在12 h、24 h、48 h时采用12.5、25、50μmol/L五味子素处理HCT116细胞,细胞活力均受到抑制(P<0.05),其中50μmol/L浓度处理48 h时,细胞存活率接近50%。与对照组、FOXC1-NC组相比,FOXC1组细胞凋亡率、E-cadherin mRNA及蛋白表达水平降低,侵袭细胞数与FOXC1、MMP2、Vimentin mRNA及蛋白表达水平升高(P<0.05);与对照组比较,五味子素组细胞凋亡率、E-cadherin mRNA及蛋白表达水平升高,侵袭细胞数与FOXC1、MMP2、Vimentin mRNA及蛋白表达水平下降(P<0.05);FOXC1+五味子素组细胞凋亡率、E-cadherin mRNA及蛋白表达水平高于FOXC1组,侵袭细胞数与FOXC1、MMP2、Vimentin mRNA及蛋白低于FOXC1组,而细胞凋亡率、E-cadherin mRNA及蛋白低于五味子素组,侵袭细胞数与FOXC1、MMP2、Vimentin mRNA及蛋白高于五味子素组(P<0.05)。结论五味子素可促进CRC HCT116细胞凋亡,抑制细胞侵袭,可能通过下调FOXC1及调节其下游E-cadherin、MMP2、Vimentin mRNA及蛋白表达来发挥抑癌作用。

Objective To investigate the mechanism of schisandrin on the malignant biological behavior of colorectal cancer(CRC)cells by regulating Forkhead boxC1(FOXC1)gene.Methods HCT116 cells were treated with different concentrations(0,12.5,25,50μmol/L)of schisandrin,and cells of appropriate concentration were selected for subsequent experiments,and were divided into the control group(no treatment),FOXC1-NC group(transfection negative FOXC1 mimic negative control),FOXC1 group(transfection FOXC1),schisandrin group(add 50μmol/L schisandrin),FOXC1+schisandrin group(transfected with FOXC1 and added 50μmol/L schisandrin),Flow cytometry to detect HCT116 cell apoptosis,transwell test to detect cell invasion ability,realtime-PCR to detect FOXC1,e-cadherin,matrix metalloproteinase 2(MMP2),vimentin mRNA expression,western blot to detect FOXC1,MMP2,e-cadherin,vimentin protein expression level.Results Compared with 0μmol/L,when HCT116 cells were treated with 12.5,25,50μmol/L schisandrin at 12 h,24 h,and 48 h,the cell viability was inhibited(P<0.05),of which the concentration of 50μmol/L was treated At 48 h,the cell survival rate was close to 50%.Compared with the control group and FOXC1-NC group,the apoptosis rate,e-cadherin mRNA and protein expression levels in FOXC1 group de-creased,and the number of invasive cells and FOXC1,MMP2,vimentin mRNA and protein expression levels increased(P<0.05);compared with the control group,the apoptosis rate,e-cadherin mRNA and protein expression levels in the schisandrin group increased,and the number of invasive cells and FOXC1,MMP2,vimentin mRNA and protein expression levels decreased(P<0.05);FOXC1+schisandrin group cells apoptosis rate,e-cadherin mRNA and protein expression levels were higher than FOXC1 group,the number of invasive cells and FOXC1,MMP2,vimentin mRNA and protein were lower than FOXC1 group,while apoptosis rate,e-cadherin mRNA and protein were lower than schisandrin group,the number of invasive cells and FOXC1,MMP2,vimentin mRNA and protein were higher than those in the schisandrin group(P<0.05).Conclusions Schisandrin can promote the apoptosis of CRC HCT116 cells and inhibit cell invasion.It may play a tumor suppressor effect by down-regulating FOXC1 and regulating its downstream e-cadherin,MMP2,vimentin mRNA and protein expression.