麦冬皂苷D通过调控miR-519d-3p/EIF4E表达对肝癌细胞增殖、迁移、侵袭的实验研究

Ophiopogonin D inhibits proliferation, migration, and invasion of hepatocellular carcinoma cells by regulating miR-519d-3p/EIF4E expression

背景麦冬皂苷D(ophiopogonin D,OPD)是中药麦冬提取物中重要的单体成分且具有抗癌作用,但是否具有抗肝细胞癌(hepatocellular carcinoma,HCC)作用还未知.本研究假设OPD能够通过上调miR-519d-3p表达进而下调真核细胞翻译起始因子4E(eukaryotic translation initiation factor 4E,EIF4E)表达发挥抗HCC作用.目的探讨OPD对HCC细胞增殖、迁移和侵袭的影响及可能的作用机制.方法培养HCC细胞HepG2和MHCC97,不同浓度(2.5μmol/L、5μmol/L、10μmol/L)的OPD作用48 h后,四甲基噻唑蓝染色法(methylthiazoletrazolium,MTT)检测细胞增殖,Transwell检测细胞迁移和侵袭,实时荧光定量PCR(real-time quantitative PCR,RT-qPCR)检测细胞中miR-519d-3p和EIF4E mRNA表达,Western blot检测细胞周期蛋白D1(CyclinD1)、p21、基质金属蛋白酶2(matrix metalloproteinase-2,MMP-2)、MMP-9和EIF4E蛋白表达.双荧光素酶报告基因实验验证miR-519d-3p和EIF4E之间关系.转染miR-519d-3p mimics、si-EIF4E构建miR-519d-3p过表达或EIF4E表达抑制的HepG2和MHCC97细胞,RT-qPCR检测细胞中miR-519d-3p表达或Western blot检测EIF4E蛋白表达验证转染效率.MTT、Transwell、Western blot分别检测过表达miR-519d-3p或抑制EIF4E表达对HepG2和MHCC97细胞增殖、迁移和侵袭,及CyclinD1、p21、MMP-2和MMP-9蛋白表达的影响.结果与对照组比,OPD组HepG2细胞抑制率显著升高(P<0.05),迁移和侵袭数显著降低(P<0.05),HepG2细胞中CyclinD1、MMP-2和MMP-9蛋白表达显著降低(P<0.05),p21蛋白表达显著升高(P<0.05),miR-519d-3p表达显著升高(P<0.05),EIF4E mRNA和蛋白表达显著降低(P<0.05),且呈浓度依赖性.miR-519d-3p在HepG2细胞中靶向负调控EIF4E表达.miR-519d-3p过表达或抑制EIF4E表达均可抑制HepG2细胞增殖、迁移和侵袭.抑制miR-519d-3p表达部分逆转了OPD对HepG2细胞增殖、迁移和侵袭的抑制作用.结论OPD可能通过调控miR-519d-3p/EIF4E表达抑制HCC细胞的增殖、迁移和侵袭.

BACKGROUND Ophiopogonin D(OPD)is an important monomer component in Chinese traditional medicine.Ophiopogon extract has anti-cancer effects,but it is unknown whether it has anti-liver cancer effects.We hypothesized that OPD could have anti-liver cancer activity by up-regulating the expression of miR-519d-3p and then down-regulating the expression of eukaryotic translation initiation factor 4E(EIF4E).AIM To investigate the effects of OPD on proliferation,migration,and invasion of hepatocellular carcinoma cells and the possible mechanism involved.METHODS HepG2 and MHCC97 cells were cultured for 48 h after treatment with different concentrations(2.5,5,and 10μmol/L)of OPD.Methylthiazoletrazolium(MTT)assay was used to detect cell proliferation,Transwell assay was used to detect cell migration and invasion,real-time quantitative PCR(RT-qPCR)was used to detect the levels of miR-519d-3p and EIF4E mRNA in HepG2 cells,and Western blot was used to detect the expression levels of CyclinD1,p21,matrix metalloproteinase(MMP)-2,MMP-9,and EIF4E proteins.Dual luciferase reporter gene assay was used to validate the relationship between miR-519d-3p and EIF4E.To obtain HepG2 or MHCC97 cells with miR-519d-3p overexpression or EIF4E knockdown,miR-519d-3p mimic or si-EIF4E was transfected into HepG2 or MHCC97 cells.Then,RT-qPCR was used to detect the level of miR-519d-3p expression in HepG2 or MHCC97 cells and Western blot was performed to detect the level of EIF4E protein to verify the transfection efficiency.MTT assay,Transwell assay,and Western blot were used to detect the effects of overexpression of miR-519d-3p or inhibition of EIF4E on cell proliferation,migration,and invasion as well as the expression of CyclinD1,p21,MMP-2,and MMP-9 proteins.RESULTS Compared with control cells,the rates of reduced growth of HepG2 cells in the OPD groups were significantly increased(P<0.05),cell migration and invasion were significantly decreased(P<0.05),the levels of CyclinD1,MMP-2,and MMP-9 proteins were significantly decreased(P<0.05),p21 protein expression was significantly increased(P<0.05),miR-519d-3p expression was significantly increased(P<0.05),and the levels of EIF4E mRNA and protein were significantly decreased(P<0.05).MiR-519d-3p negatively regulated EIF4E expression in HepG2 cells.Overexpression of miR-519d-3p or inhibition of EIF4E inhibited the proliferation,migration,and invasion of HepG2 cells.Inhibition of miR-519d-3p expression partially reversed the inhibitory effect of OPD on the proliferation,migration,and invasion of HepG2 cells.CONCLUSION OPD inhibits the proliferation,migration,and invasion of hepatoma cells possibly by regulating the expression of miR-519d-3p/EIF4E.