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Inhibition of plasminogen activator inhibitor‐1 expression by siRNA in rat hepatic stellate cells
Author(s) -
Hu PingFang,
Zhu YingWei,
Zhong Wei,
Chen YueXiang,
Lin Yong,
Zhang Xin,
Yin Chuan,
Yue HaiYan,
Xie WeiFen
Publication year - 2008
Publication title -
journal of gastroenterology and hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.214
H-Index - 130
eISSN - 1440-1746
pISSN - 0815-9319
DOI - 10.1111/j.1440-1746.2008.05485.x
Subject(s) - small interfering rna , hepatic stellate cell , microbiology and biotechnology , transfection , plasminogen activator , plasminogen activator inhibitor 1 , gene silencing , extracellular matrix , cell growth , rna interference , cell culture , biology , rna , biochemistry , endocrinology , gene , genetics
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.