Suppression of tissue inhibitor of metalloproteinase-1 by recombinant adeno-associated viruses carrying siRNAs in hepatic stellate cells.
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.
Cong
Min
M
Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, PR China.
Liu
Tianhui
T
Wang
Ping
P
Xu
Yong
Y
Tang
Shuzhen
S
Wang
Baoen
B
Jia
Jidong
J
Liu
Yong
Y
Hermonat
Paul L
PL
You
Hong
H
D000818:Animals; D000229:Dependovirus / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D020868:Gene Silencing; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D055166:Hepatic Stellate Cells / Q000201:enzymology* / Q000821:virology*; D053509:Matrix Metalloproteinase 13 / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D051381:Rats; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics* / Q000378:metabolism / Q000627:therapeutic use*; D014161:Transduction, Genetic
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D019715:Tissue Inhibitor of Metalloproteinase-1; D053509:Matrix Metalloproteinase 13