Wang Yang, Su Dong Wei, Gao Li, Ding Gui Ling, Ni Can Rong, Zhu Ming Hua
Department of Pathology, Changhai Hospital, The Second Military Medical University, Shanghai, 200433, China.
Cell Biochem Biophys. 2014 Jul;69(3):487-94. doi: 10.1007/s12013-014-9822-x.
The aim of this study is to investigate the influence of Lenti-EGFP-NeuroD-miR, RNAi lentiviral expression vector, on the expression level of NeuroD and migration, and invasion of PANC-1 cell line. PANC-1 cells were cultured and cotransfected with Lenti-EGFP-NeuroD-miR and Lenti-GFP. The infection rate of lentivirus was determined by fluorescence. The interfering effection by the expression of NeuroD mRNA in PANC-1 cells was analyzed by real-time PCR after transfected. Biological behavior of PANC-1 cells transinfected was observed, and the migration and invasion were studied by transwell assay. Intrapancreatic allografts model in nude mice was established to observe the effects of NeuroD on tumorigenesis, tumor growth, and invasion in vivo. The expression of NeuroD mRNA decreased significantly after RNAi lentivirus transinfecting PANC-1 cell. The cell's migration and invasion ability decreased obviously as soon as down regulate of NeuroD in PANC-1 cells. Comparing with control group, the tumors were smaller in size and the invasiveness was inhibited after 8 weeks intrapancreatic allografts in nude mice. Lenti-EGFP-NeuroD-miR transfected into PANC-1 cells shows a stable, effective, and especial blocking expression of NeuroD in mRNA level. The RNAi of lentiviral vector target NeuroD can reduce the migration and invasion abilities of PANC-1 cells.
本研究旨在探讨RNAi慢病毒表达载体Lenti-EGFP-NeuroD-miR对PANC-1细胞系中NeuroD表达水平以及迁移和侵袭能力的影响。培养PANC-1细胞，并将Lenti-EGFP-NeuroD-miR与Lenti-GFP共转染。通过荧光测定慢病毒的感染率。转染后，采用实时PCR分析PANC-1细胞中NeuroD mRNA表达的干扰效果。观察转染后PANC-1细胞的生物学行为，并通过Transwell实验研究其迁移和侵袭能力。建立裸鼠胰腺内同种异体移植模型，观察NeuroD对体内肿瘤发生、肿瘤生长和侵袭的影响。RNAi慢病毒转染PANC-1细胞后，NeuroD mRNA表达显著降低。PANC-1细胞中NeuroD表达下调后，其迁移和侵袭能力明显下降。与对照组相比，裸鼠胰腺内同种异体移植8周后，肿瘤体积较小，侵袭性受到抑制。转染到PANC-1细胞中的Lenti-EGFP-NeuroD-miR在mRNA水平上对NeuroD表现出稳定、有效且特异的阻断表达。慢病毒载体靶向NeuroD的RNAi可降低PANC-1细胞的迁移和侵袭能力。
