微小RNA-340对肝细胞癌细胞转移和侵袭力的影响

Effect of microRNA-340 on migration and invasion of hepatocellular carcinoma cell

目的探讨高表达微小RNA-340(miR-340)对肝细胞癌(HCC)细胞转移和侵袭力的影响。方法采用LipofectamineTM2000试剂盒进行miR-340转染,Transwell侵袭实验评价侵袭能力,实时荧光定量聚合酶链反应(qPCR)测定HCC细胞株miR-340、E-钙黏蛋白、N-钙黏蛋白、波形蛋白、基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶-9(MMP-9)的mRNA水平,酶联免疫吸附测定法(ELISA)测定MMP-2和MMP-9蛋白水平,用蛋白质免疫印迹(Western blot)检测核转录因子κB1(NF-κB1)蛋白水平。结果与HepG2相比miR-340在HCC细胞系中表达水平降低,高表达miR-340的HepG2细胞、MHCC-97L细胞侵袭能力下降。高表达miR-340的HepG2细胞和MHCC-97L细胞E-钙黏蛋白mRNA表达上调,N-钙黏蛋白mRNA水平下调,波形蛋白mRNA水平下调。miR-340使HepG2细胞和MHCC-97L细胞中MMP-2和MMP-9的表达下降;与HCC细胞系比较,高表达miR-340的HepG2细胞NF-κB1表达量下降40%、MHCC-97L细胞下降66%,差异有统计学意义(P<0.05)。生物信息学分析预示NF-κB1是miR-340的潜在靶基因且萤光素酶报告分析又进一步证实了miR-340可以直接作用于NF-κB1的3′端非编码区。结论 miR-340在抑制细胞迁移、侵袭和上皮细胞间质转化中起着重要的作用,提示miR-340表达水平降低是HCC发生的重要环节,基于miR-340的治疗方法可用于HCC诊治。

Objective To explore the effect of microRNA-340(miR-340)on the migration and invasion of human hepatocelluar carcinoma(HCC)cells.Methods The miR-340 was transiently transfected into HCC cells using LipofectamineTM2000 reagent.Using Transwell invasion assay to evaluate the invasion force of HCC cells,The mRNA level of miR-340,E-cadherin,N-cadherin,Vimentin,MMP-2 and MMP-9 in HCC cells were determined by real-time quantitative polymerase chain reaction(qPCR).The protein levels of MMP-2 and MMP-9 were detected by ELISA,Western blot were used to analysis the expression of NF-κB1 protein.Results Compared with FTE187,the expression of microRNA-340 in HCC cell lines was decreased.overexpression of microRNA-340 inhibited the invasion ability of HepG2 and MHCC-97 Lcells.The expression of E-cadherin was up-regulated in HepG2 and MHCC-97 Lcells with high expression of microRNA-340,the level of N-cadherin and vimentin was down-regulated in these cells.The expression of MMP-2 and MMP-9 in HepG2 and MHCC-97 Lcells was decreased by microRNA-340.Compared with miR-NC cells,the expression of NF-kappa B1 in HepG2 cells with high expression of microRNA-340 was decreased by 40% and that of MHCC-97 Lcells by 66%,the difference was statistically significant(P<0.05).Bioinformatics analysis predicted that the NF-κB1 was a potential target gene of miR-340.Luciferase reporter assay further confirmed that miR-340 could directly target the 3′UTR of NF-κB1.Conclusion miR-340 plays an important role in inhibiting migration,invasion and EMT,suggesting that miR-340 expression is one of the important mechanisms of HCC,miR-340-based therapy can be used to treat HCC.