橄榄苦苷通过调控ELK-3抑制人肝癌细胞上皮-间质转化的机制研究

Mechanism study of oleuropein inhibiting epithelial-mesenchymal transition of human liver cancer cells

目的探讨橄榄苦苷与ELK-3以及人肝癌细胞上皮-间质转化(epithelial-mesenchymal transition,EMT的关系及其作用机制。方法采用MTT法检测不同浓度橄榄苦苷(50、100、200、400、800μmol/L)对HepG2细胞活力的影响,筛选合适的作用浓度。Western blot法检测HepG2细胞中ELK-3、p38以及EMT标志物E-cadherin、vimentin蛋白表达水平。Transwell迁移小室检测HepG2细胞侵袭能力。结果MTT法检测结果显示,与空白对照组相比,不同浓度(50、100、200、400、800μmol/L)的橄榄苦苷作用于HepG2细胞后,细胞存活率均下降,且有浓度依赖性,差异有统计学意义(P<0.05),选取HepG2细胞存活率接近50%的200μmol/L橄榄苦苷进行后续实验。与人正常肝细胞(LO2细胞)相比,HepG2细胞中的ELK-3蛋白表达水平显著升高,差异有统计学意义(P<0.05);橄榄苦苷(200μmol/L)处理HepG2细胞后ELK-3、vimentin和p38磷酸化水平均显著降低,E-cadherin蛋白表达水平显著升高,差异有统计学意义(P<0.05)。Transwell侵袭实验显示,细胞侵袭24 h后,空白对照组成功转移的细胞数为(61±4)个,橄榄苦苷组(200μmol/L)成功转移的细胞数为(13±1)个,HepG2细胞侵袭能力明显下降,差异有统计学意义(P<0.05)。结论橄榄苦苷通过调控ELK-3/p38抑制人肝癌细胞EMT的发生。

Objective To investigate the relationship and mechanism of oleuropein with ElK-3 and epithelialmesenchymal transition(EMT)of human liver cancer cells.Methods MTT assay were used to investigate the effects of different concentrations of oleuropein(50,100,200,400,800μmol/L)on the viability of HepG2 cells,and the appropriate concentration was screened.The protein levels of ELK-3,p38 and EMT markers E-cadherin,vimentin in HepG2 cells were detected by Western blot.The invasion ability of HepG2 cells was detected by Transwell migration chamber.Results MTT assay showed that compared with blank control group,the survival rate of HepG2 cells treated with different concentrations of oleuropein(50,100,200,400,800μmol/L)decreased in a concentration-dependent manner,the difference was statistically significant(P<0.05).200μmol/L oleuropein with a survival rate of nearly 50%HepG2 cells was selected for follow-up experiments.Compared with human normal hepatocyte LO2 cells,the expression level of ELK-3 protein in HepG2 cells was significantly increased,the difference was statistically significant(P<0.05).And the expression levels of ELK-3,vimentin and p38 phosphorylation were significantly decreased and the expression level of E-cadherin were significantly increased after treatment with 200μmol/L oleuropein(P<0.05).Transwell invasion assay showed that 24 hours after cell invasion,the number of successfully transferred cells in blank control group was(61±4),and(13±1)in oleuropein group(200μmol/L).The invasion ability of HepG2 cells was significantly decreased,the difference was statistically significant(P<0.05).Conclusions Oleuropein inhibits the occurrence of EMT in human liver cancer cells by regulating ELK-3/p38.