Yang Xuhui, Xia Tian, Yu Weihua, Lu Xiaofang, Xiang Peng, He Feng
Guangdong Women and Children's Hospital, Guangzhou, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2012 Jan;32(1):7-13.
To study the effect of lentivirus-mediated RNA interference (RNAi) of nestin on the metastatic potential of human melanoma cell line UACC903.
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.
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 (P<0.05).
Lentivirus-mediated nestin RNAi can specifically inhibit nestin expression to cause decreased cell migration and invasiveness of human melanoma cell line UACC903.
研究慢病毒介导的巢蛋白RNA干扰（RNAi）对人黑色素瘤细胞系UACC903转移潜能的影响。
构建针对人巢蛋白mRNA编码区的RNAi慢病毒载体（巢蛋白-RNAi-LV）和另一个含无义序列的对照载体。将载体转染至UACC903细胞，通过逆转录-聚合酶链反应（RT-PCR）、免疫荧光分析和蛋白质印迹法检测细胞中巢蛋白的表达。分别采用Transwell小室法和划痕实验评估转染后UACC903细胞的侵袭能力和迁移能力。采用荧光分析检测细胞中E-钙黏蛋白、N-钙黏蛋白和β-连环蛋白的表达。
成功构建了慢病毒载体巢蛋白-RNAi-LV。与对照载体相比，巢蛋白-RNAi-LV导致巢蛋白mRNA和蛋白表达明显降低，UACC903细胞迁移能力下降，细胞黏附及侵袭能力降低（P<0.05）。
慢病毒介导的巢蛋白RNAi可特异性抑制巢蛋白表达，导致人黑色素瘤细胞系UACC903细胞迁移和侵袭能力下降。
