Expression of tissue inhibitor of metalloproteinase-1 and matrix metalloproteinase 13 in rat hepatic stellate cell infected by adeno-associated virus carrying small interfering RNA

Objective To construct recombinant adeno-associated virus(AAV)carrying small interfering RNA(siRNA)of tissue inhibitor of metalloproteinase-1(TIMP-1)and investigate the long-term effect of TIMP-1 gene RNA interference on rat hepatic stellate cell(HSC)-T6 cells and the influence on the expression of matrix metalloproteinase(MMP)13 in vitro.Methods U6 promotor followed by the annealing siRNA which had the strongest suppression 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.After infection of this recombinant AAV into rat HSC-T6 cells and selection by G418,real-time PCR after reverse transcription and Western blot were performed to detect the transcription and expression level of TIMP-1 gene and MMP13 gene in HSC-T6 cells at 4 weeks and 12 weeks.Results The results of PCR,restrictive enzyme digestion and gene sequencing confirmed that the pdl6-95/siRNA-TIMP-1/neo had been reconstructed successfully.After it had been packed in 293 cell to form rAAV/siRNA-TIMP-1/neo and infected into HSC-T6,the transcription and expression level of TIMP-1 in HSC-T6 cells which were infected by the rAAV/siRNA-TIMP-1/neo were suppressed dramatically compared with mock control and normal HSC-T6 cells(P0.01),and transcription and expression level of MMP13 in HSC-T6 cells infected by the rAAV/siRNA-TIMP-1/neo were increased significantly compared with mock control and normal HSC-T6 cells(P0.01).Conclusion RNA interference can cause suppression of TIMP-1 gene in rat HSC,and when this function combined with AAV infection it can suppress the specific gene expression for long time.