Department of Respiratory Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, P.R. China.
Int J Mol Med. 2012 Dec;30(6):1343-8. doi: 10.3892/ijmm.2012.1137. Epub 2012 Sep 20.
The aim of this study was to construct a eukaryotic expression vector for a small interfering RNA (siRNA) targeting the neural precursor cell expressed, developmentally downregulated 9 (NEDD9) gene, and to investigate the effects of RNA interference (RNAi) on NEDD9 expression in human lung adenocarcinoma A549 cells. We used the siRNA design and analysis software to determine the target oligonucleotides according to the sequence of NEDD9 mRNA available in GenBank. Four siRNA sequences were obtained, and the corresponding cDNAs were synthesized and inserted into the pRNAT-CMV3.2 plasmid to construct the recombinant plasmids. These were transformed into the E. coli strain DH5α. The plasmids, after identification by PCR and DNA sequencing, were transfected into the A549 cell line via the liposome method. NEDD9 mRNA and protein in the cells were determined by fluorescence quantitative RT-PCR (FQ-PCR) and western blotting, respectively. The pRNAT-CMV3.2-transfected plasmid was used as a control. Four recombinant plasmids were identified by PCR and sequence analysis, which contained the correct insertion of the designed sequences in the plasmids. FQ-PCR and western blotting showed substantially decreased mRNA and protein expression of the NEDD9 gene in the transfected cells, compared with the control group. In conclusion, the recombinant plasmids expressing the siRNA targeting the NEDD9 gene were successfully constructed, and the siRNA expression vectors inhibited the expression of NEDD9 in A549 cells.
本研究旨在构建靶向神经前体细胞表达的发育下调基因 9（NEDD9）的小干扰 RNA（siRNA）的真核表达载体，并探讨 RNA 干扰（RNAi）对人肺腺癌细胞 A549 中 NEDD9 表达的影响。我们使用 siRNA 设计和分析软件，根据 GenBank 中提供的 NEDD9 mRNA 序列确定了靶寡核苷酸。获得了四个 siRNA 序列，并合成了相应的 cDNA 并插入到 pRNAT-CMV3.2 质粒中，构建了重组质粒。这些质粒转化到大肠杆菌 DH5α 菌株中。通过 PCR 和 DNA 测序鉴定质粒后，通过脂质体法将其转染到 A549 细胞系中。通过荧光定量 RT-PCR（FQ-PCR）和 Western blot 分别测定细胞中的 NEDD9 mRNA 和蛋白质。pRNAT-CMV3.2 转染的质粒用作对照。通过 PCR 和序列分析鉴定了四个重组质粒，其中包含质粒中设计序列的正确插入。FQ-PCR 和 Western blot 显示，与对照组相比，转染细胞中 NEDD9 基因的 mRNA 和蛋白质表达明显降低。综上所述，成功构建了靶向 NEDD9 基因的 siRNA 表达载体，该 siRNA 表达载体抑制了 A549 细胞中 NEDD9 的表达。
