NEDD8抑制剂MLN4924抑制肝细胞癌恶性表型的研究

Experimental study on the inhibition of malignant phenotype of hepatocellular carcinoma by NEDD8 inhibitor MLN4924

目的研究NEDD8抑制剂MLN4924对肝细胞癌增殖、迁移侵袭等恶性表型的影响及其分子机制。方法以人肝癌细胞株Hep3B为研究对象,将细胞分为3组,分别为MLN4924组(使用NEDD8抑制剂MLN4924处理)、阴性对照组(等体积DMSO处理)和空白对照组(不作任何处理)。Western blot方法检测NEDD8蛋白及其介导的neddylation的水平及E-cadherin和N-cadherin蛋白的表达。CCK-8实验检测细胞增殖。Transwell实验和划痕实验检测细胞迁移侵袭能力。结果与阴性对照组相比,MLN4924组NEDD8蛋白表达量增加,而NEDD8介导的neddylation减少,并且细胞的增殖、迁移侵袭能力均明显降低,差异具有统计学意义(P<0.01)。与阴性对照组或空白对照组相比,MLN4924组细胞伪足消失,缩小变圆,呈上皮细胞形态,同时E-cadherin蛋白表达明显升高,N-cadherin蛋白表达明显降低,差异具有统计学意义(P<0.01)。结论MLN4924通过抑制NEDD8介导的neddylation从而抑制肝癌细胞增殖、迁移侵袭等恶性表型,同时抑制EMT发生。

Objective To explore the effects of NEDD8 inhibitor MLN4924 on the malignant phenotypes of hepatocellular carcinoma cells such as proliferation,migration and invasion,and then explore the related underlying molecular mechanisms.Methods Human hepatocellular carcinoma cell lines Hep3B were divided into three groups:MLN4924 group(treated with MLN4924,an inhibitor of NEDD8),negative control group(treated with DMSO)and blank control group(no treatment).The expression levels of NEDD8,neddylation,E-cadherin and N-cadherin were detected by Western blot.The cell proliferation was observed by CCK-8 assays.The cell migration and invasion abilities were observed by Transwell and wound healing assays.Results The expression of NEDD8 protein in MLN4924 group was higher than that in negative control group,while the neddyylation level of NEDD8 was lower(all P<0.01).And the cell proliferation,migration and invasion abilities in MLN4924 group were lower than those in negative control group(P<0.01).At the same time,the cells were round and showed epithelial cell morphology,and pseudopodia disappeared in MLN4924 group.In addition,after treated with MLN4924,E-cadherin protein expression was increased,while N-cadherin protein expression was decreased(P<0.01).Conclusion MLN4924 could inhibit the neddylation level mediated by NEDD8 in hepatocellular carcinoma cells,effectively inhibit malignant phenotypes such as cell proliferation,migration and invasion,and inhibit EMT signal pathway.