银椴苷通过调控miR-218/NEDD9轴影响肝癌细胞增殖、迁移、侵袭和凋亡

Tiliroside Affects the Proliferation,Invasion,Migration and Apoptosis of Hepatocellular Carcinoma Cells Through Regulating miR-218/NEDD9 Axis

目的探讨银椴苷对肝癌细胞生物学行为的影响和分子机制。方法采用不同浓度(0.1、0.2、0.4、0.8、1.6 mmol/L)的银椴苷分别处理肝癌细胞Huh748h,通过四甲基偶氮唑蓝(MTT)法、流式细胞术、Transwell实验检测细胞活力、凋亡率以及迁移、侵袭能力。实时荧光定量PCR(qRT-PCR)检测miR-218和神经元细胞表达发育下调基因9(NEDD9)mRNA的表达水平,蛋白质印迹(Westernblot)检测NEDD9蛋白表达水平。双荧光素酶报告基因实验和Westernblot验证miR-218和NEDD9的靶向调控关系。将miR-218抑制物(anti-miR-218)转染Huh7细胞,检测干扰miR-218表达联合银椴苷处理对Huh7细胞活力、凋亡、迁移和侵袭的影响。结果与空白组比较,银椴苷处理组Huh7细胞活力、迁移和侵袭数、NEDD9mRNA和蛋白表达显著降低,凋亡率、miR-218表达显著升高(均P<0.05)。miR-218可与NEDD9直接结合。过表达miR-218后NEDD9表达水平降低(均P<0.05),干扰miR-218后NEDD9表达水平升高(均P<0.05)。干扰miR-218表达可逆转银椴苷对Huh7细胞增殖、迁移侵袭、凋亡的影响(均P<0.05)。结论银椴苷可诱导肝癌细胞凋亡,抑制其增殖、迁移和侵袭,其机制可能是通过上调miR-218/NEDD9轴实现的。

Objective To investigate the effects and molecular mechanisms of tiliroside on the biological behavior of hepatocellular carcinoma cells.Methods Hep7 cells were treated with different concentrations of tiliroside(0.1,0.2,0.4,0.8,1.6 mmol/L)for 48 h.Cell viability,apoptotic rate,migration and invasion ability were detected by MTT assay,flow cytometry and Transwell assay,respectively.Real-time quantitative PCR(qRT-PCR)was used to detect the expression level of miR-218 and neuronal cell expression down-regulated gene 9(NEDD9)mRNA.Western blotting was used to detect the expression levels of NEDD9 protein.Dual luciferase reporter gene assay and Western blotting were performed to verify the targeted and regulatory relationship between miR-218 and NEDD9.Huh7 cells were transfected with miR-218 inhibitor(anti-miR-218),and the effects of interfering with miR-218 combined with tiliside treatment on the viability,apoptosis,migration and invasion of Huh7 cells were investigated by the above method.Results Compared with the blank group,the cell viability,migration numbers,invasion numbers and NEDD9 expression in tiliroside treatment group were significantly decreased,apoptosis rate and miR-218 expression were significantly increased(P<0.05).miR-218 directly bound to NEDD9.NEDD9 expression was significantly decreased after overexpression of miR-218(P<0.05),while NEDD9 expression was significantly increased after interference with miR-218(P<0.05).Interference with miR-218 could reverse the effects of tiliroside on the proliferation,migration and apoptosis of Huh7 cells(P<0.05).Conclusion Tiliroside could induce cell apoptosis and inhibit the proliferation,migration,invasion of hepatocellular carcinoma cells,and the mechanism might be achieved by up-regulating the miR-218/NEDD9 axis.