[Comparison between the suppression of tissue inhibitor of metalloproteinase-1 gene expression by recombinant adeno-associated virus carrying antisense RNA and small interfering RNA (siRNA) of TIMP-1 in rat hepatic stellate cells].
OBJECTIVE
Elevated tissue inhibitor of metalloproteinase-1 (TIMP-1) expression contributes to excess extracellular matrix in liver fibrosis. This study was designed to construct two recombinant adeno-associated viruses (AAV) carrying antisense RNA and small interfering RNA (siRNA) of TIMP-1 (rAAV/ANTI-TIMP-1/neo and rAAV/siRNA-TIMP-1/neo), and then to compare the suppression of TIMP-1 gene expression on rat hepatic stellate cell (HSC)-T6 cells infected by these two types of viruses in vitro.


METHODS
Antisense RNA amplified by rat HSC-T6 and U6 promoter followed by the annealing siRNA were cloned into the AAV vector (pdl6-95/neo) and packed in 293 cells to construct the recombinants rAAV/ANTI-TIMP-1/neo and rAAV/siRNA-TIMP-1/neo. Rat HSC-T6 cells were infected with these recombinant AAVs and selected by using G418, and real-time PCR after reverse transcription and Western blot were performed to detect the transcription and expression level of TIMP-1 gene in these cells.


RESULTS
The results of PCR, restrictive enzyme digestion and gene sequencing confirmed that the pdl6-95/ANTI-TIMP-1/neo and pdl6-95/siRNA-TIMP-1/neo had been reconstructed successfully. After they had been packed in 293 cells to form rAAV/ANTI-TIMP-1/neo and rAAV/siRNA-TIMP-1/neo, they were used to infect HSC-T6. Thirty days after the infection, the transcription level of TIMP-1 in HSC-T6 cells infected by rAAV/siRNA-TIMP-1/neo decreased dramatically compared with the mock control and normal HSC-T6 cells (P less than 0.01), and the expression level of TIMP-1 gene in HSC-T6 cells decreased significantly (60%), while the transcription and expression level of TIMP-1 in HSC-T6 cells infected by rAAV/ANTI-TIMP-1/neo had no significant difference with mock control and normal HSC-T6 cells (P more than 0.05).


CONCLUSIONS
RNA interference can exert a suppression of TIMP-1 gene in rat HSC, and when this function combines with AAV infection, it can suppress the specific gene expression for a long time by chromosomal integration.
Cong
Min
M
Liver Research Center, Beijing Friendship Hospital, Capital University of Medical Sciences, Beijing 100050, China.
Wang
Ping
P
Liu
Tian-Hui
TH
Xu
Yong
Y
Lu
Yan
Y
Tang
Shu-Zhen
SZ
Liu
Xiao-Ming
XM
Wang
Bao-En
BE
Jia
Ji-Dong
JD
You
Hong
H
D000818:Animals; D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics; D005822:Genetic Vectors; D055166:Hepatic Stellate Cells / Q000378:metabolism*; D016372:RNA, Antisense / Q000235:genetics; D034741:RNA, Small Interfering; D051381:Rats; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism*
D016372:RNA, Antisense; D034741:RNA, Small Interfering; D019715:Tissue Inhibitor of Metalloproteinase-1