Effect of Eukaryotic Expression Plasmid for Cyclooxygenase-2 Specific siRNA on Growth of HT-29 Cells in vitro

Objective To investigate the effect of the eukaryotic expression plasmid of specific small interfering RNA (siRNA)against COX-2 gene on the COX-2 expression and growth of human colon cancer HT-29 cells. Methods The COX-2 siRNA template DNA sequence for short hairpin RNA (shRNA) was designed and synthesized. The recombinant plasmid (pshCOX-2) was transfected into HT-29 cells. The effect of the recombinant plasmid on the COX-2 expression of human colon cancer HT-29 cells was detected by RT-PCR and Western blot. Changes of the cell growth activity in response to transfected plasmid were evaluated by MTT assay and FCM. The methods of RIA and ELISA were respectively used to estimate the content of PGE2 and VEGF in supernatant. Results It was confirmed by restrictive enzyme digestion and sequence analysis that the recombinant plasmid was cloned and the aim sequence was obtained. The COX-2 expression of HT-29 cells was inhibited at mRNA and protein levels 72 hours after transfected with the recombinant pshCOX-2. The growth of HT-29 cells was inhibited and the apoptotic ratio was significantly highly in COX-2 siRNA transfected group than those in control groups(P<0.05). The content of PGE2 and VEGF in supernatant decreased significantly by transfecting pshCOX-2. Conclusion COX-2 siRNA expression plasmid pshCOX-2 successfully constructed can inhibit the expression of COX-2 gene，proliferation and synthesis of PGE2 and VEGF of HT-29 cells.