微小RNA-4478下调鼠双微体-2对结肠癌细胞增殖、迁移、侵袭的影响

Effect of miR-4478 down-regulating MDM2 on proliferation, migration and invasion of colon cancer cells and its molecular mechanism

目的探讨微小RNA-4478(miR-4478)对结肠癌细胞增殖、迁移、侵袭的作用并阐明相关机制。方法实时定量聚合酶链式反应(PCR)检测miR-4478在结肠癌组织中的表达。结肠癌SW1116细胞转染miR-4478 mimics及阴性对照后,分别采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)和小室(Transwell)法检测细胞的增殖、迁移及侵袭。预测miR-4478的靶基因,双荧光素酶报告基因实验验证靶基因。结肠癌SW1116细胞转染miR-4478 mimics或抑制物后,蛋白质免疫印迹(Western blot)法检测细胞靶基因表达。靶基因过表达验证细胞增殖、迁移及侵袭。Western blot法检测细胞周期蛋白D1(Cyclin D1)、基质金属蛋白酶2(MMP-2)、P21、钙粘附蛋白E(E-cadherin)蛋白表达。结果相对于癌旁组织,miR-4478在结肠癌组织中的表达显著降低[(0.84±0.07)比(0.26±0.02),t=48.461,P<0.05];转染miR-4478 mimics可显著抑制细胞增殖、迁移及侵袭(P<0.001)。miR-4478可负向调控MDM2表达(P<0.001)。miR-4478过表达可抑制由MDM2过表达导致的结肠癌细胞增殖、迁移及侵袭(P<0.001)。结论miR-4478通过抑制MDM2表达而抑制结肠癌细胞增殖、迁移及侵袭。

Objective To investigate the effects of microRNA-4478(miR-4478)on proliferation,migration and invasion of colon cancer cells and to elucidate the relevant mechanisms.Methods Real-time quantitative PCR was used to detect the expression of miR-4478 in colon cancer tissues.Colon cancer SW1116 cells were transfected with miR-4478 mimics and negative controls,and cell proliferation,migration and invasion were detected by MTT and Transwell methods,respectively.The target gene of miR-4478 was predicted,and the dual luciferase reporter gene experiment was used to verify the target gene.After colon cancer SW1116 cells were transfected with miR-4478 mimics or inhibitors,the expression of target genes was detected by Western blot.Target gene overexpression confirmed cell proliferation,migration and invasion.The expressions of Cyclin D1,MMP-2,P21 and E-cadherin were detected by Western blot.Results The expression of miR-4478 was significantly reduced in colon cancer tissues[(0.84±0.07)vs.(0.26±0.02),t=48.461,P<0.05].Transfection of miR-4478 mimics significantly inhibited cell proliferation,migration and invasion(P<0.001).miR-4478 could negatively regulate MDM2 expression(P<0.001).Overexpression of miR-4478 inhibited colon cancer cell proliferation,migration and invasion caused by overexpression of MDM2(P<0.001).Conclusion miR-4478 inhibits colon cancer cell proliferation,migration and invasion by inhibiting MDM2 expression.