OBJECTIVE: To construct a novel combination gene vector pcDNA3.1(-) VEGF-siRNA/yCDglyTK and to evaluate its expression in and killing effects on human colon cancer HCT116 cells.
STUDY DESIGN: An interference plasmid pGenesilVEGF- siRNA targeting vascular endothelial growth factor (VEGF) was first constructed, from which siRNA expression cassette (including U6 promoter) was amplified by PCR and subcloned into a double suicide gene vector pcDNA3.1(-) CV-yCDglyTK to construct pcDNA3.1(-) VEGF-siRNA/yCDglyTK. The recombinant plasmids were identified by enzyme digestion and sequencing. By using calcium phosphate nanoparticles as carriers, HCT116 cells were transfected with interference plasmid, double suicide gene plasmid, and combination gene plasmid. The expressions of target genes were verified by RT-PCR and Western blot. Sensitivities of the transfected cells toward 5-flucytosine (5-FC) were detected by MTT assay.
RESULTS: As proved by enzyme digestion and sequencing, pcDNA3.1(-) VEGF-siRNA/yCDglyTK was constructed successfully. RT-PCR and Western blot showed that fused suicide gene was expressed and VEGF gene expression was downregulated. After being treated with 5-FC, the combination gene-transfected cells had significantly lower survival rate than those of the other groups.
CONCLUSION: The results preliminarily demonstrated that VEGF-targeted RNA interference and double suicide gene system exerted a synergistic effect.
[1]Jilin Univ, China Japan Union Hosp, Dept Gastrocol Surg, Changchun, Peoples R China.
[2]Jilin Canc Hosp, Dept Thyroid Surg, Changchun, Peoples R China.
(8.0+极速模式)