摘要
目的探讨RNA干扰Slug基因表达对胰腺癌转移的抑制作用。方法用脂质体转染法将表达siRNA-Slug的真核表达载体pGenesil-1-Slug-siRNA(pSlug-siRNA)和空载体pGenesil-1-Neg-siRNA(pNeg-siRNA)导入PANC-1细胞,G418筛选阳性细胞,获得稳定转染的阳性克隆。观察siRNA-Slug对Slug表达的沉默及对胰腺癌细胞体外侵袭转移的抑制作用,采用实时荧光定量聚合酶链反应(RT-PCR)技术检测不同处理组细胞中Slug mRNA的表达水平,Western-blot测定蛋白表达水平。建立不同组裸鼠原位胰腺癌模型,观察Slug沉默对裸鼠原位胰腺癌转移的抑制作用。结果pSlug-siRNA组细胞内Slug mRNA及Slug蛋白的表达被有效沉默。重组细胞基底膜(Matrigel)侵袭实验显示,pSlug-siRNA组、空白对照组、pNeg-siRNA组穿透基底膜细胞数分别为(35±10)、(228±71)、(219±72)个/高倍镜(×200),pSlug-siRNA组明显低于空白对照组和pNeg-siRNA组,差异分别有统计学意义(P<0.05)。裸鼠体内实验表明,pSlug-siRNA组的肿瘤转移结节、肝脏转移结节、腹水明显减少。结论RNA干扰Slug基因表达对胰腺癌细胞侵袭转移有抑制作用,Slug可能成为胰腺癌基因治疗的新靶点。
Objective To investigate the inhibitory effects of slug-a zinc finger transcription factor anti-sense plasmid on the invasion of pancreatic cancer cell lines in vitro. Methods pGenesil-1-Slun siRNA （pSlug-siRNA） and pGenesil-1-Neg-siRNA（pNeg- siRNA） were transfected into PANC-1 cells by Lipofectamine 2000 methods, stable transfected clony was chosen through G418. PANC-1 cells were divided into three groups：transfected pSlug-siRNA,transfected pNeg-siRNA and control group without transfection. The inhibitory effects of anti-sense Slug were also detected by Transwell motility assay and Matrigel invasion assay. RT- PCR and Western-blot were used to observe slug protein and mRNA in cells in vitro and in vivo. Results Slug was inhibited in PANC-1 cells in pSlug-siRNA groups. The invasion activity （35 ± 10）, （219 ± 72） and （228 ± 71） of antisense Slug transfectant cells were significantly decreased compared with those of control cells （P〈0.05） respectively. The number of turnout nodules,me tabasis nodurles in liver was less than that of controls,and less abdominal dropsy was seen in pSlug-siRNA groups. Conclusion Slug is possibly a potential target for cancer gene therapy blocking invasion and metastasis in human pancreatic cancer.
出处
《重庆医学》
CAS
CSCD
北大核心
2010年第5期513-515,F0002,F0003,共5页
Chongqing medicine
基金
海南省自然科学研究基金资助(809043)
