沉默法尼基转移酶对人涎腺腺样囊性癌细胞迁移、侵袭及上皮间充质转化的作用

Effects of silencing farnesyltransferase on the migration,invasion,and epithelial-mesenchymal transition of salivary adenoid cystic carcinoma cells

目的通过体外实验探讨法尼基转移酶(FTase)对涎腺腺样囊性癌SACC-LM和SACC-83细胞迁移侵袭及上皮间充质转化(EMT)的作用及其机制。方法针对人FTase基因序列设计构建3条小干扰RNA(si-RNA),采用处于对数生长期的SACC-LM及SACC-83细胞,经脂质体瞬时转染技术抑制细胞FTase表达,分别命名为FTase-siRNA-1组、FTase-siRNA-2组、FTase-siRNA-3组,同时设置阴性对照组(NC-siRNA),空白对照组(仅加入转染试剂)。采用实时荧光定量逆转录聚合酶链反应检测FTase、HRAS的mRNA表达,选择沉默效率最高的FTase-siRNA进行后续实验;蛋白质免疫印迹法检测FTase、HRAS、蛋白激酶B(AKT)、磷酸化AKT、p65、磷酸化p65(Ser563)、上皮钙依赖黏附蛋白、波形蛋白、基质金属蛋白酶9(MMP-9)的蛋白表达以及HRAS膜蛋白表达;Transwell小室及细胞划痕实验检测细胞的侵袭和迁移能力。结果与空白对照组及阴性对照组相比,FTase-siRNA-1组mRNA及蛋白相对表达量均降低(P<0.05),HRASmRNA和总蛋白表达的差异无统计学意义(P>0.05),HRAS膜蛋白相对表达量降低(P<0.05),上皮钙依赖黏附蛋白相对表达量升高(P<0.05),波形蛋白相对表达量降低(P<0.05),MMP-9蛋白相对表达量降低(P<0.05),RAS/PI3K/AKT/核因子-κB信号通路相关蛋白AKT、p65相对表达量的差异无统计学意义(P>0.05),但磷酸化AKT、磷酸化p65蛋白相对表达量降低;与空白及阴性对照组相比,FTase-siRNA-1组细胞侵袭及迁移能力显著下降(P<0.05)。结论体外沉默FTase可有效抑制人涎腺腺样囊性癌细胞SACC-LM和SACC-83的侵袭、迁移能力,其作用可能是通过干扰HRAS膜蛋白的定位,调控RAS/PI3K/AKT/核因子-κB信号通路,介导EMT而实现。

Objective This study aimed to investigate the effects of farnesyltransferase(FTase)on the migration,invasion,and epithelial-mesenchymal transition(EMT)of SACC-LM and SACC-83 cells in salivary adenoid cystic carcinoma and determine the relative mechanism.Methods Three small interfering RNA(siRNA)sequences were designed and constructed based on the human FTase gene sequence.The SACC-LM and SACC-83 cells in the logarithmic growth period were used,and the expression of FTase was suppressed by liposomal transient transfection.The tested cells were categorized as the FTase-si RNA-1,FTase-si RNA-2,and FTase-si RNA-3 groups.Both negative control group(NC-si RNA)and blank control group(only transfection reagent was added)were set.The m RNA expression of FTase and HRAS was detected by quantitive real-time polymerase chain reaction,and the silencing efficiency was determined.The expression levels of FTase,HRAS,protein kinase B(AKT),phospho-AKT,p65,phospho-p65(Ser563),E-cadherin,vimentin,matrix metalloproteinase(MMP)-9 protein,and HRAS membrane protein were detected by Western blot.Transwell assay and wound healing assay were used to detect the invasion and migration abilities of cells.Results The relative expression of FTase m RNA and protein in the FTase-si RNA-1 group decreased compared with those in the control group(P<0.05).HRAS m RNA and total protein expression had no significant difference(P>0.05),and the relative expression of HRAS membrane protein decreased(P<0.05).The relative expression of E-cadherin increased(P<0.05),vimentin decreased(P<0.05),and MMP-9 decreased(P<0.05).There was no significant difference in the relative expression levels of the RAS/PI3K/AKT/nuclear factor-κB signaling pathway-related proteins AKT and p65(P>0.05),but the relative expression levels of phospho-AKT and phospho-p65 decreased.The invasion and migration ability of the FTasesi RNA-1 group significantly decreased compared with that in the control group(P<0.05).Conclusion Silencing FTase in vitro could effectively inhibit the invasion and migration of SACC-LM and SACC-83 cells by interfering with the localization of the HRAS membrane protein and regulating the RAS/PI3K/AKT/nuclear factor-κB signaling pathway to mediate EMT.