Journal of gastroenterology and hepatology · 2008
Inhibition of plasminogen activator inhibitor-1 expression by siRNA in rat hepatic stellate cells.
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Hu Ping-Fang, Zhu Ying-Wei, Zhong Wei, Chen Yue-Xiang, Lin Yong, Zhang Xin, Yin Chuan, Yue Hai-Yan, Xie Wei-Fen
Аннотация
BACKGROUND AND AIM: 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.
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.
Предметные рубрики MeSH
ActinsAnimalsCarbon TetrachlorideCell LineCell ProliferationCollagen Type ICollagen Type IIIDisease Models, AnimalDown-RegulationHepatic Stellate CellsLiverLiver Cirrhosis, ExperimentalMalePlasminogen Activator Inhibitor 1RNA InterferenceRNA, MessengerRNA, Small InterferingRatsRats, Sprague-DawleyTime FactorsTissue Inhibitor of Metalloproteinase-1TransfectionTransforming Growth Factor beta