Construction of a novel vector expressing Survivin-shRNA and fusion suicide gene yCDglyTK and its application in inhibiting proliferation and migration of colon cancer cells

Abstract

Despite progress achieved in cancer chemotherapy in recent decades, adverse effects remain a limiting factor for a number of patients with colorectal cancer, suggesting the requirement for novel therapeutic strategies. Gene therapy appears to be a promising strategy for treating cancer. The present study aimed to investigate the anti-tumor effect of a combined gene therapy, using Survivin downregulation by RNAi and a fusion suicide gene yCDglyTK therapy system. A triple-gene vector expressing Survivin-targeted small hairpin RNA (Survivin-shRNA) and fusion suicide gene yCDglyTK was constructed, and administered to HCT116 cells. Survivin expression decreased significantly and yCDglyTK fusion gene expression was confirmed by both reverse transcription-quantitative polymerase chain reaction and western blot analysis. Introduction of Survivin-shRNA into yCDglyTK/prodrug system eradicated colon cancer cells and induced apoptosis more effectively. Furthermore, this therapeutic system is able to inhibit the migration of HCT116 cells. These results indicate that the recombinant plasmid may serve as a novel gene therapy approach to treat colorectal carcinoma.

Keywords: RNAi; Survivin; combination gene therapy; suicide gene.

Figure 1.

Construction of the pYr1.1-hTERTp-yCDglyTK-shSurvivin2 plasmid. (A) Expression of EGFP in HCT116 cells and human fibroblasts by transfection with pYr1.1-hTERTp. Top image demonstrates the EGFP-positive expression of HCT116 cells by fluorescence microscopy; bottom image demonstrates the negative expression of EGFP in fibroblasts (magnification, ×200). (B) Construction scheme of a novel plasmid pYr1.1-hTERTp-yCDglyTK-shSurvivin2. EGFP, enhanced green fluorescent protein; TK, thymidine kinase; yCD, yeast cytosine deaminase; hTERT, human telomerase reverse transcriptase; Survivin-shSur, Survivin-targeted small hairpin RNA.

Figure 2.

Inhibition of Survivin mRNA and protein expression by transfection with CDTK-shSur. Representative Survivin mRNA and protein expression were analyzed by (A) RT-qPCR and (B) western blot analysis, respectively. β-actin was used as an internal control. Lane 1, parent HCT116; lane 2, HCT/CDTK; lane 3, HCT/CDTK-shSur. (C) Density of each band was measured, densities of Survivin were normalized against corresponding β-actin signals, and relative intensities were expressed in arbitrary units where the intensity of parent HCT116 cells was set to 100%. *P<0.01 vs. all other groups. The data are expressed as mean + standard deviation from three independent experiments. CDTK, fusion suicide gene involving yeast CD gene and herpes simplex virus thymidine kinase gene; shSur, Survivin-targeted small hairpin RNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Figure 3.

Expression of yCDglyTK by transfection with CDTK-shSur. Representative yCDglyTK mRNA and protein expression were analyzed by (A) RT-qPCR and (B) western blot analysis, respectively. β-actin was used as an internal control. Lane 1, parent HCT116; lane 2, HCT/CDTK; lane 3, HCT/CDTK-shSur. (C) Representative yCDglyTK protein expression detected by immunofluorescence assays. Magnification, ×200. CDTK or yCDglyTK, fusion suicide gene involving yeast CD gene and herpes simplex virus thymidine kinase gene; shSur, Survivin-targeted small hairpin RNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Figure 4.

CTDK-shSur/prodrug system induces cytotoxicity and apoptosis. (A) Growth curves of HCT116 cells (transfected and untransfected) following administration of 5-FC and GCV. HCT116 cells (transfected and untransfected) maintained in culture medium containing 5-FC (200 µg/ml) and GCV (16 µg/ml). At 24, 48, 72 and 96 h, cells of each group were subjected to MTT assays. Cell growth curves were plotted with culture time as the horizontal axis and OD570 as the vertical axis. (B) HCT116 cells (transfected and untransfected) were treated with prodrugs (5-FC and GCV) for 48 h, and the apoptosis rate of each group was measured by flow cytometry. *P<0.05 vs. HCT group and ^#P<0.05 vs. HCT/CDTK group. Data are presented as mean + standard deviation. CDTK, fusion suicide gene involving yeast CD gene and herpes simplex virus thymidine kinase gene; shSur, Survivin-targeted small hairpin RNA; 5-FC, 5-fluorocytosine; GCV, ganciclovir; OD, optical density.

Figure 5.

CDTK-shSur inhibits cancer cell migration. (A) Representative images of parent HCT116 and HCT/CDTK-shSur cells in wound healing assay (magnification, ×100). (B) Quantitative analysis of the migration was made by measuring the migration distance 24 h after scratching. *P<0.01 vs. HCT. Data are presented as mean + standard deviation. CDTK, fusion suicide gene involving yeast CD gene and herpes simplex virus thymidine kinase gene; shSur, Survivin-targeted small hairpin RNA.