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Suppression of tissue inhibitor of metalloproteinase-1 by recombinant adeno-associated viruses carrying siRNAs in hepatic stellate cells.
- Source :
-
International journal of molecular medicine [Int J Mol Med] 2009 Nov; Vol. 24 (5), pp. 685-92. - Publication Year :
- 2009
-
Abstract
- Elevated tissue inhibitor of metalloproteinase (TIMP)-1 expression contributes to excess production of extracellular matrix in liver fibrosis. However, there are few studies on sustained suppression of TIMP-1. We aimed to construct a recombinant adeno-associated virus (AAV) carrying small interfering RNAs (siRNAs) of TIMP-1 and investigate the long-term effects of RNA interference upon the TIMP-1 gene in rat hepatic stellate cells (HSCs). Five siRNA oligomers targeting rat TIMP-1 were designed and transfected into HSCs. A U6 promoter followed by the siRNA which had the strongest suppression effect was cloned into the AAV vector and packed into 293 cells to construct the recombinant AAV/siRNA-TIMP-1/neo. After infecting HSCs with this recombinant AAV, the transcription and expression levels of the TIMP-1 and matrix metalloproteinase-13 (MMP-13) genes were detected at 4 and 12 weeks. Three of the five designed siRNA oligomers had a suppressing effect on TIMP-1 expression in rat HSCs within 72 h. The transcription and expression levels of TIMP-1 were suppressed significantly (P<0.05) following recombinant AAV/siRNA1-TIMP-1/neo infection and lasted 12 weeks. TIMP-1 expression in rAAV/siRNA1-TIMP-1/neo-infected HSCs was suppressed by 60% after four weeks and 90% after twelve weeks when compared to the control recombinant AAV/neo and uninfected HSCs. Furthermore, the transcription and protein expression levels of MMP-13, the main substrate of TIMP-1, were elevated by approximately 40% at twelve weeks in rAAV/siRNA-TIMP-1/neo-infected HSCs. RNA interference exerts suppressive effect on the TIMP-1 gene in cultured HSCs for a longer time when a recombinant AAV is utilized as the gene delivery vector.
- Subjects :
- Animals
Gene Expression Regulation, Enzymologic
Gene Silencing
Genetic Vectors genetics
Matrix Metalloproteinase 13 metabolism
RNA, Messenger genetics
RNA, Messenger metabolism
Rats
Tissue Inhibitor of Metalloproteinase-1 metabolism
Transduction, Genetic
Dependovirus genetics
Genetic Therapy
Hepatic Stellate Cells enzymology
Hepatic Stellate Cells virology
RNA, Small Interfering metabolism
Tissue Inhibitor of Metalloproteinase-1 genetics
Tissue Inhibitor of Metalloproteinase-1 therapeutic use
Subjects
Details
- Language :
- English
- ISSN :
- 1791-244X
- Volume :
- 24
- Issue :
- 5
- Database :
- MEDLINE
- Journal :
- International journal of molecular medicine
- Publication Type :
- Academic Journal
- Accession number :
- 19787203
- Full Text :
- https://doi.org/10.3892/ijmm_00000280