基于miR-208a-3p探究萝卜硫素对肝癌细胞增殖、侵袭和迁移的影响

Effect of sulforaphane on proliferation,invasion and migration of liver cancer cells based on miR-208a-3p

目的探讨萝卜硫素对肝癌细胞增殖、侵袭和迁移的影响及其作用机制。方法用10、20、40μmol/L萝卜硫素处理人正常肝细胞L02、肝癌HepG2细胞48 h,通过CCK-8法筛选合适的萝卜硫素浓度用于后续实验;将HepG2细胞分为对照组和萝卜硫素(20μmol/L)组,CCK-8法检测细胞增殖;Transwell测定细胞迁移与侵袭。设置对照组、miR-208a-3p模拟物(miR-208a-3p mimic)组及其阴性对照(mimic NC)组、miR-208a-3p抑制物(miR-208a-3p inhibitor)组及其阴性对照(inhibitor NC)组、真核翻译起始因子4E(eukaryotic translation initiation factor 4E,EIF4E3)过表达物(pcDNA-EIF4E3)组及其阴性对照(pcDNA)组、EIF4E3小干扰RNA(si-EIF4E3)组及其阴性对照(si-NC)组、pcDNA-EIF4E3+mimic NC组和pcDNA-EIF4E3+miR-208a-3p mimic组,利用LipofectamineTM 2000转染试剂盒对HepG2细胞进行转染,转染48 h后,各组分别加入20μmol/L萝卜硫素处理24 h,qRT-PCR检测HepG2细胞中miR-208a-3p表达;CCK-8法检测细胞增殖;Transwell测定细胞迁移与侵袭;双荧光素酶报告基因检测实验验证miR-208a-3p与EIF4E3靶向关系;Western blotting检测细胞中EIF4E3蛋白表达。结果与对照组比较,萝卜硫素组HepG2细胞在24、48 h的A450值、侵袭及迁移细胞数目显著降低(P<0.05);HepG2细胞中miR-208a-3p呈高表达,EIF4E3蛋白呈低表达;miR-208a-3p靶向负调控EIF4E3的表达;下调miR-208a-3p或过表达EIF4E3可增强萝卜硫素对HepG2细胞增殖、侵袭及迁移的抑制作用,而过表达miR-208a-3p或沉默EIF4E3则呈相反趋势;同时过表达miR-208a-3p和EIF4E3不会影响萝卜硫素对HepG2细胞增殖、侵袭及迁移的抑制作用。结论萝卜硫素可能通过下调miR-208a-3p进而上调EIF4E3的表达来抑制HepG2细胞增殖、侵袭及迁移。

Objective To investigate the effect and mechanism of sulforaphane on proliferation,invasion and migration of liver cancer cells.Methods Human normal liver cells L02 and hepatocarcinoma HepG2 cells were treated with 10,20,40μmol/L sulforaphane for 48 h,and the appropriate sulforaphane concentration was screened by CCK-8 method for subsequent experimental studies;HepG2 cells were divided into control group and sulforaphane(20μmol/L)group,CCK-8 method was used to detect cell proliferation;Transwell was used to measure cell migration and invasion.HepG2 cells were divided into control group,miR-208a-3p mimic group,mimic group,miR-208a-3p inhibitor group,inhibitor NC group,pcDNA-eukaryotic translation initiation factor 4E(EIF4E3)group,pcDNA group,si-EIF4E3 group,si-NC group,pcDNA-EIF4E3+mimic NC group and pcDNA-EIF4E3+miR-208a-3p mimic group,HepG2 cells were transfected with LipofectamineTM 2000 transfection kit,after 48 h of transfection,each group was treated with 20μmol/L sulforaphane for 24 h.qRT-PCR was used to detect the expression of miR-208a-3p in HepG2 cells;CCK-8 method was used to detect cell proliferation;Transwell method was used to measure cell migration and invasion;Dual luciferase reporter gene detection experiment was used to verify the targeting relationship between miR-208a-3p and EIF4E3;Western blotting was used to detect the EIF4E3 protein expression in cells.Results Compared with control group,A450 value,number of invasion and migration cells of HepG2 cells in sulforaphane group were significantly reduced(P<0.05);miR-208a-3p in HepG2 cells was highly expressed and EIF4E3 protein was low expressed;miR-208a-3p targeted and negatively regulated the expression of EIF4E3;Down-regulation of miR-208a-3p or over-expression of EIF4E3 could enhance the inhibitory effects of sulforaphane on HepG2 cell proliferation,invasion and migration,while overexpression of miR-208a-3p or silence of EIF4E3 showed the opposite trend;Overexpression of miR-208a3p and EIF4E3 at the same time would not affect the inhibitory effects of sulforaphane on HepG2 cell proliferation,invasion and migration.Conclusion Sulforaphane may down-regulate miR-208a-3p to up-regulate the expression of EIF4E3,thereby inhibiting the proliferation,invasion and migration of HepG2 cells.