翠云草总黄酮通过调控miR-3662表达抑制喉鳞癌TU177细胞增殖、迁移和侵袭的机制研究

Mechanism of Total Flavonoids from Selaginella uncinata Inhibiting the Proliferation,Migration and Invasion of Laryngeal Squamous Cell Carcinoma TU177 Cells by Regulation of miR-3662

目的探讨翠云草总黄酮(total flavonoids from Selaginella uncinata,TFS)对喉鳞癌TU177细胞增殖、迁移和侵袭的影响及可能机制.方法体外培养TU177细胞,分为对照组、不同剂量(5、15、25μg/mL)TFS组,CCK-8法和克隆形成实验检测细胞增殖,划痕实验检测细胞迁移,Transwell检测细胞侵袭,Western印迹检测细胞中上皮型钙粘蛋白(E-Cadherin)、神经型钙粘蛋白(N-Cadherin)表达,RT-qPCR法检测miR-3662表达.收集35例喉鳞癌患者的癌组织及癌旁组织,RT-qPCR法检测组织中miR-3662表达.另取TU177细胞,分为miR-3662组、miR-NC组、25μg/mL TFS+anti-miR-3662组和25μg/mL TFS+anti-miR-NC,CCK-8法和克隆形成实验检测细胞增殖,划痕实验检测细胞迁移,Transwell检测细胞侵袭,Western印迹检测细胞中E-Cadherin和N-Cadherin蛋白表达.结果与对照组比较,不同剂量TFS组TU177细胞抑制率升高(P<0.05),克隆形成数、划痕愈合率和侵袭数及细胞中N-Cadherin蛋白表达降低(P<0.05),E-Cadherin蛋白和miR-3662表达升高(P<0.05),且呈剂量依赖性.喉鳞癌组织中miR-3662的表达较癌旁组织显著降低(P<0.05).与miR-NC组比较,miR-3662组喉鳞癌TU177细胞抑制率和细胞中E-Cadherin蛋白表达升高(P<0.05),克隆形成数、迁移和侵袭数及细胞中N-Cadherin蛋白表达均降低(P<0.05).与25μg/mL TFS+anti-miR-NC组比较,25μg/mL TFS+anti-miR-3662组TU177细胞抑制率和细胞中E-Cadherin蛋白表达降低(P<0.05),克隆形成数、迁移和侵袭数及细胞中N-Cadherin蛋白表达均升高(P<0.05).结论翠云草总黄酮可抑制喉鳞癌TU177细胞增殖、迁移和侵袭,其作用机制可能与上调细胞中miR-3662表达有关.

Objectivee To investigate the effect of total flavonoids from Selaginella uncinata(TFS)on the proliferation,migration and invasion of laryngeal squamous cell carcinoma TU177 cells and its possible mechanism.Methods TU177 cells were cultured in vitro and were divided into control group,TFS-5μg/mL,-15μg/mL,-25μg/mL groups,miR-3662 group,miR-NC group,25μg/mL TFS+anti-miR-3662 group and 25μg/mL TFS+anti-miR-NC group.CCK-8 method and colony formation assay were used to detect cell proliferation.Scratch assay was used to detect cell migration.Transwell assay was used to detect cell invasion.Western blotting was used to detect the protein expression levels of epithelial cadherin(E-Cadherin)and neural cadherin(NCadherin)in cells,and RT-qPCR method was used to detect the expression level of miR-3662.Results Compared with the control group,the cell inhibition rate of TFS groups was increased(P<0.05),but the number of colonies,the wound healing rate and the number of invasions,and the expression level of N-Cadherin protein were decreased(P<0.05),while the expression levels of E-Cadherin protein and miR-3662 were increased(P<0.05),all in a dose-dependent manner.The expression level of miR-3662 in the laryngeal squamous cell carcinoma tissues was significantly lower than that in the adjacent tissues(P<0.05).Compared with the miR-NC group,the cellinhibition rate and the expression level of E-Cadherin protein in the miR-3662 group were increased(P<0.05),but the number of colonies,the wound healing rate and the number of invasions,and the expression level of N-Cadherin protein were decreased(P<0.05).Compared with the 25μg/mL TFS+anti-miR-NC group,the cell inhibition rate and the expression level of E-Cadherin protein in the 25μg/mL TFS+anti-miR-3662 group were decreased(P<0.05),but the number of colonies,the wound healing rate and the number of invasions,and the expression level of NCadherin protein were increased(P<0.05).Conclusion Total flavonoids from Selaginella uncinata could inhibit the proliferation,migration and invasion of laryngeal squamous cell carcinoma TU177 cells,and its mechanism may be related to the up-regulation of miR-3662 expression in the cells.