上调微RNA-3178表达对肝癌血管内皮细胞侵袭、迁移的影响

摘要

Objective To explore the effect of invasion and migration of hepatocellular carcinoma (HCC) endothelial cells (TECs) affected by overexpression of microRNA-3178 (miR-3178) through the transfection of miR-3178 mimic.Methods Real-time polymerase chain reaction (Real-time PCR) was used to identify differential expression of miR-3178 in normal hepatic sinusoidal endothelial cells (HSECs) and HCC TECs.Furthermore,HCC TECs were divided into 3 groups:control (CON) group,miRNA-3178 upregulation (Mimics,up-regulation of miR-3178 expression was achieved using miR-3178 mimics transfected into HCC TECs) group and negative control (NC,negative control sequence was transfected into HCC TECs) group.RT-PCR was used to detect expression of miR-3178 in HCC TECs before and after transfection.Transfection efficiency was observed by using an inverted fluorescence microscope.HCC TECs invasionand migration were measured by matrigel invasion and transwell migration assay.EGR3 protein expression of HCC TECs were identified by Western blotting analysis.EGR3 mRNA expression of HCC TECs were identified by RT-PCR analysis.Results The results of RT-PCR showed that miR-3178 was significantly down-regulated in HCC TECs compared to HSECs (P ＜0.05),and expression of miR-3178 was significantly increased after the transcienttransfection (P ＜ 0.05).The transfection efficiency in HCC TECs was morethan 90％.Number of migrated and invaded cells and in miR-3178 group was significantly less than those in other groups.Target gene prediction software showed EGR3 was a possible candidate target.Transfection of miR-3178 mimic significantly decreased the mRNA and protein expression levels of EGR3.Conclusion MiR-3178 was downregulated in HCC TECs and overexpression of miR-3178 can specifically inhibit migration and invasion of HCC TECsin vitro through inhibiting EGR3 expression,thus,miR-3178 might be a critical targeted therapy strategv for HCC.%目的 通过合成及转染微RNA(miRNA)-3178模拟物(mimics),研究上调miR-3178表达对肝癌血管内皮细胞(HCC TECs)侵袭、迁移的影响.方法 实时定量聚合酶链反应(RT-PCR)验证肝窦内皮细胞(HSECs)和HCC TECs中miR-3178的差异表达.实验分为三组:空白对照组(CON组)、miR-3178上调组(Mimics组,miRNA-3178 mimic转染HCC TECs)、阴性对照组(NC组,miR-3178 mimic阴性对照序列转染HCC TECs).RT-PCR验证转染前后HCC TECs中miR-3178表达.荧光显微镜观察转染效率.Transewell法检测HCC TECs侵袭及迁移.生物信息学软件预测miR-3178靶基因,蛋白印迹(Western blot)及RT-PCR验证靶基因.结果 RT-PCR结果显示HCCTECs中miR-3178表达明显低于HSECs(P＜0.05).转染后HCC TECs中miR-3178表达明显高于转染前(P＜0.05),细胞转染效率达到90％以上.细胞侵袭及迁移试验结果显示,Mimic组侵袭及迁移细胞数明显减少(P＜0.05).生物信息学软件预测EGR3可能是miR-3178的靶基因之一.Western blot及RT-PCR结果显示,上调miR-3178表达,可明显抑制HCC TECs中EGR3 mRNA及蛋白表达(P＜0.05).结论 HCC TECs中miR-3178明显低表达,上调miR-3178表达可通过抑制EGR3表达而抑制HCC TECs侵袭及迁移,靶向抑制HCC TECs中miR-3178表达可能为肝癌重要的治疗方法.