Inhibition of plasminogen activator inhibitor-1 expression by siRNA in rat hepatic stellate cells.
OBJECTIVE
The plasminogen activator/plasmin system is known to regulate the extracellular matrix turnover. The aim of this study was to detect the role of plasminogen activator inhibitor-1 (PAI-1) during liver fibrogenesis and investigate the functional effects of PAI-1 gene silencing in rat hepatic stellate cells (HSCs) using small interfering RNA (siRNA).


METHODS
Hepatic fibrosis in rats was induced through serial subcutaneously injections of CCl(4) and the expression of PAI-1 was detected by immunohistochemistry and reverse transcription-polymerase chain reaction (PCR). PAI-1 siRNA molecules were constructed and transiently transfected into HSC-T6 using the cell suspension transfection method. The pSUPER RNA interfering system was used to establish the HSC stable cell line pSUPER-shPAI. Expression of alpha-smooth muscle actin, transforming growth factor-beta, tissue inhibitor of metalloproteinases-1, and collagen types I and III were evaluated by real-time PCR. Cell proliferation and the cell cycle were determined by the methyl thiazolyl tetrazolium (MTT) method and flow cytometry. Collagen content in HSCs supernatant was evaluated by enzyme-linked immunosorbent assay.


RESULTS
The results showed that PAI-1 was upregulated during liver fibrosis, and its expression was closely correlated with the deposition of collagens. SiRNA molecules were successfully transfected into HSCs and induced inhibition of PAI-1 expression time dependently. Moreover, PAI-1 siRNA treatment downregulated alpha-smooth muscle actin, transforming growth factor-beta, tissue inhibitor of metalloproteinases-1 expression, and inhibited collagen types I and III synthesis both at the mRNA and protein level in transiently and stably transfected HSCs.


CONCLUSIONS
This study suggests a significant functional role for PAI-1 in the development of liver fibrosis and that downregulating PAI-1 expression might present as a potential strategy to treat liver fibrosis.
Hu
Ping-Fang
PF
Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, Shanghai, China.
Zhu
Ying-Wei
YW
Zhong
Wei
W
Chen
Yue-Xiang
YX
Lin
Yong
Y
Zhang
Xin
X
Yin
Chuan
C
Yue
Hai-Yan
HY
Xie
Wei-Fen
WF