摘要
目的探讨通过抑制叉头框蛋白A1(forkhead box protein A1,FOXA1)的表达来调控Notch通路对结肠癌SW480细胞增殖及侵袭的影响。方法选取河南科技大学第一附属医院2019年6月至2021年2月期间收治的45例结肠癌患者的结肠癌组织及其相应的癌旁组织,采用免疫组织化学和实时荧光定量PCR(real-time fluorescent quantitative PCR,qRT-PCR)法分别检测组织中FOXA1蛋白和mRNA表达情况。另外,将结肠癌SW480细胞分为对照组(未经处理)、FOXA1沉默对照组(shRNA-NC组,转染shRNA-NC)、sh-FOXA1组(转染sh-FOXA1)、sh-FOXA1+丙戊酸钠组(转染sh-FOXA1后添加8 mmol/L Notch通路激活剂丙戊酸钠),然后采用qRT-PCR法、MTT法、克隆形成实验及Transwell法检测各组细胞中FOXA1 mRNA表达、细胞增殖、克隆形成能力、侵袭和迁移情况;Western blot法检测各组细胞中与增殖(c-Myc、cyclinD1)、侵袭和迁移[基质金属蛋白酶(matrix metalloproteinase,MMP)9、MMP2]、上皮间充质转化(Vimentin、N-cadherin、E-cadherin)及Notch通路(Notch-1、Hes-1)相关蛋白的表达情况。结果(1)临床病例中,FOXA1蛋白和mRNA表达水平在结肠癌组织中均高于其相应的癌旁组织(蛋白:0.085±0.028比0.034±0.010,t=11.036,P<0.001;mRNA:1.62±0.34比1.00±0.09,t=11.671,P<0.001)。(2)细胞实验发现,与对照组和shRNA-NC组相比,沉默FOXA1表达后即sh-FOXA1组的增殖(细胞存活率)、克隆细胞数、迁移细胞数及侵袭细胞数均明显降低(P<0.05),相应地sh-FOXA1组细胞中与之有关的蛋白c-Myc、cyclinD1、MMP9、MMP2、Vimentin、N-cadherin、Notch-1、Hes-1蛋白表达水平均明显降低(P<0.05)、E-cadherin蛋白表达水平明显升高(P<0.05);加入Notch通路激活剂丙戊酸钠后,上述情况全部被逆转(P<0.05)。结论FOXA1在结肠癌组织和结肠癌细胞中均高表达,其有可能是通过激活Notch通路来促进SW480细胞增殖、侵袭和迁移。
Objective To investigate effect of Notch pathway regulating by inhibiting expression of forkhead box protein A1(FOXA1)on proliferation and invasion of colon cancer SW480 cells.Methods The colon cancer tissues and their corresponding paracancerous tissues of 45 patients with colon cancer admitted to the First Affiliated Hospital of Henan University of Science and Technology from June 2019 to February 2021 were selected.The immunohistochemistry and real-time fluorescent quantitative PCR(qRT-PCR)methods were used to detect the expressions of FOXA1 protein and mRNA in the tissues,respectively.In addition,SW480 cells were divided into control group(untreated),shRNA-NC group(transfected with shRNA-NC),sh-FOXA1 group(transfected with sh-FOXA1),sh-FOXA1+sodium valproate group(Add 8 mmol/L Notch pathway activator sodium valproate after transfection with sh-FOXA1).Then the qRT-PCR,MTT,clone formation test,and Transwell methods were used to detect the expressions of FOXA1 mRNA,proliferation,clonogenic ability,invasion and migration of cells in each group.Western blot method was used to detect the proliferation(c-Myc,cyclinD1),invasion and migration[matrix metalloproteinase(MMP)9,MMP2],epithelial-mesenchymal transition(Vimentin,N-cadherin,E-cadherin)and Notch pathway(Notch-1,Hes-1)related protein expressions of cells in each group.Results(1)In the clinical cases,the expression levels of FOXA1 protein and mRNA in the colon cancer tissues were higher than those in the corresponding paracancerous tissues(protein:0.085±0.028 vs.0.034±0.010,t=11.036,P<0.001;mRNA:1.62±0.34 vs.1.00±0.09,t=11.671,P<0.001).(2)In the cell experiment,compared with the control group and shRNA-NC group,the cell survival rate,and numbers of cloned cells,invasion and migrating cells were significantly reduced(P<0.05),correspondingly,the related proteins expression levels of c-Myc,cyclinD1,MMP9,MMP2,Vimentin,N-cadherin,Notch-1,Hes-1 were significantly reduced(P<0.05)and the protein expression level of E-cadherin was significantly increased(P<0.05)in the sh-FOXA1 group,which were reversed after adding the Notch pathway activator sodium valproate(P<0.05).Conclusion FOXA1 highly expresses in colon cancer tissues and colon cancer cells and it might promote the proliferation,invasion and migration of SW480 cells by activating the Notch pathway.
作者
刘可峰
王伟
范永刚
于英杰
LIU Kefeng;WANG Wei;FAN Yonggang;YU Yingjie(Department of Hepatobiliary and Pancreatic Surgery,The First Affiliated Hospital of Henan University of Science and Technology,Luoyang,Henan 471000,P.R.China)
出处
《中国普外基础与临床杂志》
CAS
2022年第4期444-449,共6页
Chinese Journal of Bases and Clinics In General Surgery
基金
国家自然科学基金资助项目(项目编号:U1804193)。
