鞘氨醇激酶1对肝癌耐药细胞生物学特性的影响

摘要

目的 研究鞘氨醇激酶1(SPK1)对人肝癌耐药细胞株BEL-FU生物学特性的影响.方法 将SPK1siRNA基因(Ad-H 1-SPK1)及野生型SPK1基因(Ad-SPK1wT)导入人肝癌耐药细胞株BEL-FU.用试剂盒检测SPK酶活性,MTT法检测其对肝癌细胞凋亡的影响,Transwell法测定肝癌细胞侵袭力,Western-blot方法检测多药耐药相关蛋白1(MRP1)表达水平的变化.结果 导入Ad-SPK1wT增强肝癌细胞SPK活性,转染SPK1 siRNA(Ad-H 1-SPK1)抑制肝癌细胞SPK活性；肝癌细胞导入SPK1后,抑制SPK特异性抑制剂N,N,-二甲基鞘氨醇(Dimethyl sphingosine,DMS)引起的肝癌细胞凋亡并促进肝癌细胞迁移和增加多药耐药蛋白( MRP1)的表达；阻断SPK1则显著促进细胞凋亡和明显抑制肝癌细胞迁移,且多药耐药蛋白(MRP1)表达量明显降低.结论 SPK1的活性与肝癌细胞的凋亡、侵袭力及多药耐药密切相关,阻断SPK1可为肝癌治疗提供新的靶点和策略.%Objective To investigate the roles of sphingosine kinasel (SPK1) in apoptosis,invasiveness and multidrug resistance of human hepatocellular carcinoma cell line BEL-FU.Methods BEL-FU cells were infected with adenovirus carrying SPK1wT gene and SPK1siRNA (Ad-H1-SPK1) gene.Their effects on biological characteristics of BEL-FU cells were evaluated by MTT,cellular SPK enzyme activity assay,Transwell Migration Technology and Western-blot,respectively.Results AdSPK1wT significantly increased SPK activity but SPK1siRNA(Ad-H1-SPK1) decreased SPK activity.Over expression of SPK1 suppressed the apoptosis induced by DMS(Dimethyl sphingosine,DMS) and enhanced migration of BEL-FU cells.The cells infected with SPK1 siRNA( Ad-H1-SPK1)significantly increased the apoptosis induced by DMS and inhibited the migration of human hepatocellular carcinoma cells.The expression of multidrug resistance-related protein (MRP1) of cells infected with SPK1siRNA (Ad-H1-SPK1) was suppressed significantly compared with the control group,while the expression of MRP1 infected with Ad- SPK1wT was enhanced.Conclusion SPK1 activity is closely associated with apoptosis、migration and multidrug resistance of human hepatocellular carcinoma cells,therefore,it may serve as a new target for HCC treatment.