Inhibition of plasminogen activator inhibitor-1 expression by siRNA in rat hepatic stellate cells

J Gastroenterol Hepatol. 2008 Dec;23(12):1917-25. doi: 10.1111/j.1440-1746.2008.05485.x. Epub 2008 Aug 28.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / metabolism
  • Animals
  • Carbon Tetrachloride
  • Cell Line
  • Cell Proliferation
  • Collagen Type I / metabolism
  • Collagen Type III / metabolism
  • Disease Models, Animal
  • Down-Regulation
  • Hepatic Stellate Cells / metabolism*
  • Hepatic Stellate Cells / pathology
  • Liver / metabolism*
  • Liver / pathology
  • Liver Cirrhosis, Experimental / chemically induced
  • Liver Cirrhosis, Experimental / genetics
  • Liver Cirrhosis, Experimental / metabolism*
  • Liver Cirrhosis, Experimental / prevention & control
  • Male
  • Plasminogen Activator Inhibitor 1 / genetics
  • Plasminogen Activator Inhibitor 1 / metabolism*
  • RNA Interference*
  • RNA, Messenger / metabolism
  • RNA, Small Interfering / metabolism*
  • Rats
  • Rats, Sprague-Dawley
  • Time Factors
  • Tissue Inhibitor of Metalloproteinase-1 / metabolism
  • Transfection
  • Transforming Growth Factor beta / metabolism

Substances

  • Actins
  • Collagen Type I
  • Collagen Type III
  • Plasminogen Activator Inhibitor 1
  • RNA, Messenger
  • RNA, Small Interfering
  • Tissue Inhibitor of Metalloproteinase-1
  • Transforming Growth Factor beta
  • smooth muscle actin, rat
  • Carbon Tetrachloride