Prohibitin1基因沉默对人黑色素瘤细胞铺展、迁移及侵袭的影响

摘要

Objective To investigate the effects of prohibitin 1 (PHB)silencing on spreading,migration and inva-sion of melanoma A375 cells by RNA interference (RNAi).Methods The A375 cells were divided into the control group and experimental group.The cells in the control group were cultured in DMEMcontaining 10%FBS,while the cells in the experimental group were treated with Lipofectamine2000 and PHB siRNA.After incubation for 24 hours,RT-PCR and Western blotting were used to detect the mRNA and protein expression of PHB in transfected A375 cells.Spreading experi-ment was performed for assessing the cell spreading at 30,60 and 120 min.The migration and invasion abilities of A375 cells were detected by Scratch and Transwell invasion assay,respectively.Results The mRNA and protein expression lev-els of PHB in the experimental group were lower than those in the control group (all P<0.01).Compared with the control group,the spreading cells in the experimental group were less at 30,60 and 120 min (all P<0.01).The migration area in the experimental group was significantly less than that in the control group at 12 and 24 h after wounding healing (P<0.01).Meanwhile,the invasive ability of the experimental group was less than that of the control group (P<0.01).Con-clusion PHB gene silencing can effectively inhibit the spreading,migration and invasion of melanoma cells.%目的：探讨RNA干扰（RNAi）沉默抗增殖蛋白Prohibitin1（PHB）基因对人恶性黑色素瘤A375细胞铺展、迁移和侵袭的影响。方法利用RNAi 技术，将A375细胞分为对照组和观察组，对照组给予含10％FBS 的DMEM培养，观察组加入脂质体Lipofectamine2000和PHB siRNA，均孵育24 h。采用RT-PCR法检测两组细胞中PHB mRNA的表达，Western blot法检测两组细胞中PHB蛋白的表达，铺展实验检测两组细胞在30、60、120 min铺展进程中的铺展情况，划痕实验检测两组细胞的迁移能力，Transwell侵袭实验检测两组细胞的侵袭能力。结果观察组细胞中PHB mRNA及蛋白相对表达量均低于对照组（P均＜0．01）。铺展30、60及120 min时，观察组发生铺展的相对细胞数均均少于对照组（P均＜0．01）。划痕后12、24 h，观察组细胞迁移面积均少于对照组（P均＜0．01）。观察组细胞的侵袭能力小于对照组细胞（P＜0．01）。结论 PHB基因沉默可有效抑制黑色素瘤细胞的铺展、迁移和侵袭进程。