Suppression of tissue inhibitor of metalloproteinase-1(TIMP-1) gene expression by recombinant adeno-associated virus carrying small interfering RNA(siRNA) of TIMP-1 in rat hepatic stellate cell(HSC)

Objective To coustruct recombinant adeno-associated virus(AAV)carrying siRNA of TIMP- 1 and to investigate the long-term effect of TIMP-1 gene RNA interference on rat hepatic stellate cell(HSC)-T6 cells in vitro.Methods U6 promoter together with the following annealing siRNA which had the strongest sup- pression effect were cloned into the AAV vector(pdl6-95/siRNA-TIMP-1/neo)and packed in 293 cells to construct the recombinant AAV/siRNA-TIMP-1/neo.Rat HSC-T6 cells were infected by this recombinant AAV.After G418 selection,real-time fluorescence quantitative PCR after reverse transcription and Western blot were performed to detect the transcription and expression level of TIMP-1 gene in HSC-T6 cells at 30 d and 90 d post-infection.At the same time,PCR was performed using the DNA of the infected cells as template to amplify U6 promoter.Re- sults The results of PCR,restrictive enzyme digestion and gene sequencing confirmed that the pdl6-95/siRNA- TIMP-1/neo was constructed successfully.The transcription and expression level of TIMP-1 in HSC-T6 cells infect- ed by the recombinant AAV were suppressed dramatically compared with those in mock control and normal HSC-T6 cells(P＜0.01),and the expression level of TIMP-1 protein in HSC-T6 cells decreased by 90% at 90 d and 60% at 30 d post-infection.The amplified rat U6 promoter by PCR using the DNA of infected cells as template proved that the HSC-T6 cells could continually express siRNA-TIMP-1 for 90 d after infection.Conclusion Recombinant rAAV/siRNA-TIMP-1/neo could effectively suppress the expression of TIMP-1 gene for a long time.