摘要
目的探讨CircFOXK2通过miR-588/FBXW5轴对非小细胞肺癌(NSCLC)增殖、迁移和侵袭的影响。方法实时荧光定量PCR(qRT-PCR)、Western blot分别检测NSCLC细胞A549、正常人支气管上皮细胞NHBE中CircFOXK2、miR-588、FBXW5表达;将si-NC、si-CircFOXK2、si-CircFOXK2+inhibitor NC、si-CircFOXK2+miR-588 inhibitor分别转染至A549细胞记为为si-NC组、si-CircFOXK2组、si-CircFOXK2+inhibitor NC组、si-CircFOXK2+miR-588 inhibitor组,未做任何处理的A549细胞记为NC组。双荧光素酶报告基因实验验证CircFOXK2、miR-588、FBXW5关系;Western blot检测A549细胞FBXW5、上皮钙粘附素(E-cadherin)、神经型钙粘附蛋白(N-cadherin)、波形蛋白(Vimentin)蛋白水平;CCK-8法检测A549细胞增殖情况;流式细胞术检测A549细胞凋亡;Transwell检测A549细胞侵袭、迁移数量。结果与miR-NC+WT-FOXK2组比较,miR-588 mimic+WT-FOXK2组A549细胞的荧光素酶活性显著降低(P<0.05),而miR-588 mimic+MUT-FOXK2组A549细胞的荧光素酶活性较miR-NC+MUT-FOXK2组无显著改变(P>0.05);与miR-NC+WT-FBXW5组比较,miR-588 mimic+WT-FBXW5组A549细胞的荧光素酶活性显著下降(P<0.05),miR-588 mimic+MUT-FBXW5组较miR-NC+MUT-FBXW5组A549细胞的荧光素酶活性无显著差异(P>0.05)。与NHBE细胞相比,A549细胞中CircFOXK2、FBXW5水平显著上调(P<0.05),miR-588表达量显著下调(P<0.05)。与NC组、si-NC组相比,si-CircFOXK2组A549细胞CircFOXK2表达量、FBXW5、N-cadherin、Vimentin蛋白水平、OD450值、迁移、侵袭细胞数量显著降低(P<0.05),A549细胞miR-588表达量、E-cadherin蛋白水平、细胞凋亡率显著升高(P<0.05);与si-CircFOXK2组、si-CircFOXK2+inhibitor NC组相比较,si-CircFOXK2+miR-588 inhibitor组FBXW5水平、OD450值、A549细胞迁移、侵袭细胞数量以及N-cadherin、Vimentin蛋白水平显著上升(P<0.05),miR-588表达量、A549细胞凋亡率、E-cadherin蛋白水平显著下降(P<0.05)。而miR-588下调减弱了沉默CircFOXK2抑制A549细胞增殖、迁移和侵袭的有利影响;CircFOXK2靶向调节miR-588/FBXW5轴。结论沉默CircFOXK2可能通过上调miR-588来抑制FBXW5表达,进而对NSCLC细胞增殖、迁移和侵袭产生影响。
Objective To investigate the impacts of circFOXK2 on the proliferation,migration,and invasion of non-small cell lung cancer(NSCLC)through the miR-588/FBXW5 axis.Methods Real-time fluorescence quantitative PCR(qRT-PCR)and Western blot were applied to detect the expression levels of circFOXK2,miR-588,and FBXW5 in the NSCLC cell line A549 and the normal human bronchial epithelial(NHBE)cell line.A549 cells were treated with blank control,or transfected with si-NC,si-circFOXK2,si-circFOXK2+inhibitor NC,and si-circFOXK2+miR-588 inhibitor.The relationship among CircFOXK2,miR-588 and FBXW5 was tested by the dual-luciferase reporter assay.Western blot was applied to detect the protein levels of FBXW5,E-cadherin,N-cadherin and Vimentin in A549 cells.Cell proliferation and apoptosis in A549 cells were detected by CCK-8 assay and flow cytometry,respectively.Invasive and migratory cell numbers were measured by Transwell assay.Results Compared with those co-transfected with miR-NC+WT-FOXK2,relative luciferase activity was significantly lower in A549 cells co-transfected with miR-588 mimic+WT-FOXK2(P<0.05).There was no significant difference in the relative luciferase activity between A549 cells co-transfected with miR-588 mimic+WT-FOXK2 and miR-588 mimic+MUT-FOXK2(P>0.05).Relative luciferase activity was significantly lower in A549 cells co-transfected with miR-588 mimic+WT-FBXW5 than those co-transfected with miR-NC+WT-FBXW5.No significant difference in the relative luciferase activity was detected between A549 cells co-transfected with miR-588 mimic+MUT-FBXW5 and miR-NC+MUT-FBXW5(P>0.05).Compared with those of NHBE cells,circFOXK2 and FBXW5 were significantly upregulated(P<0.05),and miR-588 was significantly downregulated in A549 cells(P<0.05).Transfection of si-circFOXK2 significantly downregulated circFOXK2,FBXW5,N-cadherin and Vimentin,reduced optical density at 450 nm wavelength(OD450)and migratory and invasive cell numbers(P<0.05),and upregulated miR-588 and E-cadherin and increased apoptotic rate in A549 cells than those treated with blank control or transfected with si-NC(P<0.05).Knockdown of miR-588 significantly reversed the regulatory effects of silenced circFOXK2 on favoring the proliferation,migration and invasion of A459 cells.CircFOXK2 targeted and regulated the miR-588/FBXW5 axis.Conclusion Silencing circFOXK2 downregulates FBXW5 by upregulating miR-588,thereby affecting the proliferation,migration,and invasion of NSCLC cells.
作者
邓科兰
刘桂霞
厉银平
谢志斌
DENG Kelan;LIU Guixia;LI Yinping(Department of Respiratory and Critical Care Medicine,the Central Hospital of Xiaogan,Hubei,Xiaogan 432000,China)
出处
《河北医药》
CAS
2024年第7期977-982,共6页
Hebei Medical Journal
基金
湖北省卫生健康委科研项目(编号:WJ2023M156)。
