紫杉醇抑制原发性肝细胞癌增殖与EMT的作用研究

Effect of Paclitaxel on the Proliferation and EMT of Hepatocellular Carcinoma

目的探究紫杉醇对肝癌细胞系增殖与上皮-间质转化(epithelial-mesenchymal transition,EMT)的作用。方法体外建立肝癌细胞系。取不同浓度的紫杉醇作用于肝癌细胞HepG2,采用四甲基偶氮唑盐(methylthiazolyldiphenyl-tetrazolium bromide,MTT)实验检测细胞增殖,流式细胞检测凋亡,采用Transwell实验检测细胞迁移和侵袭。Western blot实验检测凋亡相关蛋白和EMT标志蛋白的表达。实时荧光逆转录定量聚合酶链反应(real-time reverse transcription quantitative polymerase chain reaction,RT-qPCR)实验评估EMT mRNA相对表达量。结果不同浓度(5、10、20μmol)的紫杉醇明显地抑制了细胞增殖。凋亡检测结果表明,与对照组相比,紫杉醇(5、10、20μmol)浓度越高,细胞凋亡越强,呈浓度-时间依赖关系(P<0.05)。紫杉醇(5、10、20μmol)明显促进了细胞中Bax和Caspase-3的表达,抑制了B淋巴细胞瘤2(B-cell lymphoma 2,Bcl-2)的表达,并且呈浓度依赖效应(P<0.05)。Transwell实验的检测结果显示,5、10、20μmol的药物显著抑制了HepG2肝癌细胞的迁移和侵袭(P<0.05)。RT-qPCR实验与Western blot实验表明,紫杉醇显著促进了上皮型钙黏蛋白(E-cadherin),抑制了神经型钙黏蛋白(N-cadherin)和波形蛋白(Vimentin)的表达(P<0.05)。结论本研究发现紫杉醇对肝癌细胞的生长和EMT具有抑制作用,提示紫杉醇可成为抗肝癌的关键药物。

Objective To explore the effects of paclitaxel on the proliferation and epithelial-mesenchymal transition(EMT)of hepatoma cell lines.Methods Hepatocellular carcinoma(HCC)cell line was established in vitro.HepG2 cells were treated with different concentrations of paclitaxel.Methylthiazolyldiphenyl-tetrazolium bromide(MTT)assay was used to detect cell proliferation,flow cytometry was used to detect apoptosis,and Transwell assay was used to detect cell migration and invasion.Western blot was used to detect the expression of apoptosis related protein and EMT marker protein.Real-time reverse transcription quantitative polymerase chain reaction(RT-qPCR)assay was used to evaluate the relative expression level of EMT mRNA.Results Cell proliferation was significantly inhibited by different concentrations of paclitaxel(5,10,20μmol).The results of apoptosis detection showed that the higher the concentration of paclitaxel(5,10,20μmol),the stronger the cell apoptosis,which showed a concentration time-dependent relationship(P<0.05).Paclitaxel(5,10,20μmol)significantly promoted the expression of Bax and Caspase-3 in cells,inhibited the expression of B cell lymphoma 2(Bcl-2),and showed a concentration-dependent effect(P<0.05).Transwell assay showed that the migration and invasion of HepG2 cells(P<0.05)was significantly inhibited by 5,10 and 20μmol drugs.RT-qPCR and Western blot showed that the expression of E-cadherin was significantly promoted and the expression of N-cadherin and Vimentin was inhibited by paclitaxel(P<0.05).Conclusion Paclitaxel can inhibit the growth and EMT of hepatoma cells,which indicates that paclitaxel can become a key anti-hepatoma drug.