Construction and Identification of the RNAi Recombinant Lentiviral Vector Targeting Human DEPDC7 Gene

摘要

Human DEP domain containing 7 (DEPDC7) gene was originally found expressing high in liver tissue and low in most other tissues, but its function was largely unknown. In this study, we construct an RNA interference (RNAi) recombinant lentiviral vector particle targeting DEPDC7 in order to knockdown its gene expression in human hepatocellular carcinoma cell line HepG2. We screened three RNAi sequences targeting DEPDC7 and a scramble sequence by the aid of short hairpin RNAs (shRNA) design tools. Then, these sequences were separately cloned into the pLV-H1-EF1 alpha-puro vector to construct four lentiviral vectors (pshRNA-DEPDC7-NC, pshRNA-DEPDC7-RNAi1, pshRNA-DEPDC7-RNAi2 and pshRNA-DEPDC7-RNAi3). All of the recombinant plasmids were identified and confirmed by double digestion and DNA sequencing. After infecting HepG2 cells, the DEPDC7 mRNA and protein expression levels were examined by real-time PCR and western blot, respectively, and the gene expression was significantly down-regulated at both levels (P < 0.01). Cell motility and invasiveness were detected by Matrigel migration and invasion assay, and the results revealed that migration and invasion of HepG2 cells were significantly increased (P < 0.05). Our study showed successful construction of three lentiviral RNAi vectors targeting DEPDC7 gene and shRNA-mediated knockdown of DEPDC7 enable promotion of cell migration and invasion.