Construction of Lentivira Expression Vector of siRNA Specific for DEC1 and Identification of Its Efficiency in Human Gastric Carcinoma Cell MGC803

摘要

AIM To construct the small interfering RNA ( s iRNA ) lentiviral expression vector specific for human DEC1 gene and to evaluate its activity of inhibiting the DEC1 expression in human gastric carcinoma cell MGC803. Methods DEC1 siRNA template DNA sequences were designed and synthesized. The annealed siRNA template was inserted into pGCSIL-PUR shRNA Cloning and Expression vector. The recombinant plasmid (pGCSIL-PUR-DECl)was transformed into DHSa cell and identified by PCR and DNA sequencing. A lentiviral expression vector containing enhanced green fluorescence protein (GFP) and DEC1 small interfering RNA (siRNA) (Lenti-DEClsi), or the control siRNA (Lenti-NC) gene was constructed. We observed the rate of siRNA transfection by using fluorescence microscope.At 72h after transfection,the silencing efficiency to DEC1 gene expression were analyzed by RT-PCR and Western blot. RESULTS DNA sequencing showed Targeting siRNA oligonucleotides were correctly inserted into the pGCSIL-PUR shRNA Cloning and Expression vector without base mutation. Four days after transfection with Lenti-DEClsi (MOI: 100), the expression of the DEC1 mRNA and protein in cells significantly decreased compared with the Lenti-NC. CONCLUSION It is the first time that we silenced the DEC1 gene byhe small interfering RNA ( s iRNA ) lentiviral expression vector specific for DEC1 in an gastric adenocarcinoma cell MGC803, which lays the basis for its application in the treatment of gastric carcinoma.