慢病毒介导shRNA沉默巢蛋白表达对人黑色素瘤细胞转移特性的影响  

Effect of lentivirus-mediated nestin gene silencing on metastatic potential of human melanoma cell line UACC903 in vitro

作　　者：杨旭辉[1,2] 夏添[2] 余伟华[2] 卢晓芳[2] 项鹏[2] 何峰[3] 

机构地区：[1]广东省妇幼保健院生殖健康与不孕症科,广东广州510010 [2]中山大学中山医学院干细胞与组织工程中心,广东广州510080 [3]中山大学药学院,广东广州510006

出　　处：《南方医科大学学报》2012年第1期7-13,共7页Journal of Southern Medical University

基　　金：国家自然科学基金(30900729;81173577);广东省自然科学基金(8451008901000380)~~

摘　　要：目的应用慢病毒介导的RNA干扰技术,有效沉默黑色素瘤细胞株UACC903巢蛋白(nestin)的表达,研究其对黑色素瘤细胞转移能力的影响。方法以人nestin mRNA编码序列作为干扰靶点,以慢病毒基因PLL3.7作为质粒载体,构建靶向人nestin的shRNA表达质粒,另构建不针对任何已知mRNA的阴性对照shRNA表达质粒,分别感染UACC903细胞并流式分选获得高纯度的nestin干扰或对照组细胞。应用RT-PCR、免疫荧光及Western blotting检测不同组别nestin表达的变化。分别用Transwell侵袭试验检测跨膜细胞的数量评估各组黑色素瘤细胞的侵袭能力,划痕法检测迁移能力改变。光镜观察各组细胞形态变化,免疫荧光检测E-cadherin、N-cadherin和β-catenin在不同组别的表达变化。结果成功构建nestin shRNA慢病毒载体nestin-RNAi-LV。RT-PCR及免疫荧光结果显示干扰组UACC903细胞nestin mRNA及蛋白表达水平较对照组显著降低。nestin下调的UACC903细胞黏附、侵袭及迁移能力下降(P<0.05)。结论慢病毒介导的shRNA能有效地静默食管癌细胞nestin基因的表达,能抑制黑色素瘤细胞UACC903迁移。Objective To study the effect of lentivirus-mediated RNA interference（RNAi） of nestin on the metastatic potential of human melanoma cell line UACC903.Methods A lentiviral vector for RNAi targeting the coding region of human nestin mRNA（nestin-RNAi-LV） and another control vector containing a nonsense sequence were constructed.The vectors were transfected into UACC903 cells,and nestin expression in the cells was detected by RT-PCR,immunofluorence assay and Western blotting.The invasive ability and migration of the transfected UACC903 cells was evaluated using Transwell and scrape assay,respectively.Fluorescence assay was used to examine the expressions of E-cadherin,N-cadherin and β-catenin in the cells.Results The lentiviral vector nestin-RNAi-LV was constructed successfully.Compared with the control vector,nestin-RNAi-LV resulted in obviously reduced expression of nestin mRNA and protein,lowered migration ability of UACC903 cells,and reduced cell adhesion and invasiveness（P0.05）.Conclusion Lentivirus-mediated nestin RNAi can specifically inhibit nestin expression to cause decreased cell migration and invasiveness of human melanoma cell line UACC903.

关 键 词：RNA干扰 人黑色素瘤 巢蛋白 迁移 

分 类 号：R730.2[医药卫生—肿瘤]