OBJECTIVEThe aim of this study was to construct a recombinant plasmid carrying fusion suicide gene CDglyTK and RNA interference eukaryotic expressing vector targeting to STAT3, and to investigate the effect of double suicide gene combined with RNAi targeting to STAT3 on HCT116 and HUVEC cells in vitro.

METHODSThe CD and TK were cloned by polymerase chain reaction (PCR), and fusion gene CDglyTK was inserted into plasmid pEGFP after DNA sequence analysis, enzyme digestion and ligation. The recombinant plasmid was analyzed by PCR amplification and electrophoresis and enzyme digestion. DNA sequences containing small hairpin structure targeting to STAT3 were synthesized and inserted into the vector. The CDglyTK gene expressions in HCT116 and HUVEC cells were examined by reverse transcription-polymerase chain reaction (RT-PCR) after transfection of HCT116 and HUVEC cells. The inhibitory effect of RNA interference vector targeting to STAT3 was analyzed by RT-PCR and Western blot. The effects of 5-FC and GCV on HCT116 and HUVEC cells transfected with the recombinant plasmids were detected by MTT staining.

RESULTSThe results of restriction enzyme digestion and PCR amplification and electrophoresis showed that the recombinant pEGFP/CDglyTK was constructed correctly. The mRNA expression of gene CDglyTK was detected in HCT116 and HUVEC cells which transfected with the recombinant plasmid. The results of RT-PCR and Western blot showed that the RNA interference expression vector targeting to STAT3 effectively inhibited the expression of STAT3 in HCT116 cells. The results of MTT test showed that the inhibition ratio of group pEGFP/CDglyTK was (63.72 ± 0.64)%, significantly higher than that of control group (P < 0.05). The inhibition rate of group pEGFP/STAT3 siRNA was (47.02 ± 0.39)%, which was lower than that of group pEGFP/CDglyTK (P < 0.05), and higher than that of control group (P < 0.05). The inhibition rate of group pEGFP/CdglyTK + pEGFP/STAT3 siRNA was (85.10 ± 0.17)%, significantly higher than those of groups pEGFP/CDglyTK and group pEGFP/STAT3 siRNA (P < 0.05). Meanwhile, in HUVEC cells, the inhibition rate of group pEGFP/CDglyTK was (70.24 ± 0.33)%, significantly higher than that of the control group (P < 0.05). The inhibition rate of group pEGFP/STAT3 siRNA was (46.32 ± 0.15)%, significantly lower than that of group pEGFP/CdglyTK (P < 0.05), and higher than that of the control group (P < 0.05). The inhibition rate of group pEGFP/CdglyTK+pEGFP/STAT3 siRNA was (87.10 ± 0.24)%, significantly higher than those of groups pEGFP/CDglyTK and pEGFP/STAT3 siRNA(P < 0.05).

CONCLUSIONThe recombinant plasmids pEGFP-CDglyTK and pEGFP/STAT3 siRNA have inhibitory effect on HCT116 and HUVEC cells. The killing effects of double suicide gene combined with RNAi targeting to STAT3 are much better than those of single gene therapy.