title: Blocking of SMAD4 expression by shRNA effectively inhibits fibrogenesis of human hepatic stellate cells.
abstract: OBJECTIVE
  In this study, to clarify the SMAD4 blocking impact on fibrosis process, we investigated its down-regulation by shRNA on activated human LX-2 cell, in vitro.
  
  
  BACKGROUND
  Liver fibrosis is a critical consequence of chronic damage to the liver that can progress toward advanced diseases, liver cirrhosis and hepatocellular carcinoma (HCC). Different SMAD proteins play as major mediators in the fibrogenesis activity of hepatic stellate cells through TGF-β pathways, but the extent of SMAD4 as a co-SMAD protein remained less clear.
  
  
  METHODS
  vector expressing verified shRNA targeting human SMAD4 gene was transfected into LX-2 cells. The GFP expressing plasmid was transfected in the same manner as a control group while leptin treated cells were employed as positive controls. Subsequently, total RNA was extracted and real-time PCR was performed to measure the mRNA levels of SMAD4, COL-1A1, α-SMA, TGF-β and TIMP-1. Furthermore, trypan blue exclusion was performed to test the effect of plasmid transfection and SMAD4 shutting-down on cellular viability.
  
  
  RESULTS
  The results indicated that the expression of SMAD4was down-regulated following shRNA transfection intoLX-2 cells (P<0.001). The gene expression analysis of fibrotic genes in LX-2 cells showed that SMAD4 blocking by shRNA significantly reduced the expression level of fibrotic genes when compared to control plasmids (P<0.001). Vector expressing SMAD4-shRNA induced no significant cytotoxic or proliferative effects on LX-2 cells as determined by viability assay (P<0.05).
  
  
  CONCLUSIONS
  The results of this study suggested that knockdown of SMAD4 expression in stellate cell can control the progression of fibrogenesis through TGF-β pathway blocking.
authors.affiliation: Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Sayyad
authors.initials: S
authors.lastname: Khanizadeh
authors.affiliation: Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Mehrdad
authors.initials: M
authors.lastname: Ravanshad
authors.affiliation: Department of Bacteriology and Virology, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: SeyedYounes
authors.initials: S
authors.lastname: Hosseini
authors.affiliation: Infectious & Tropical Diseases Research Center, Hormozgan University of Medical Sciences, Bandar Abbas, Iran.
authors.forename: Parivash
authors.initials: P
authors.lastname: Davoodian
authors.affiliation: Research Center for Molecular Medicine, Hormozgan University of Medical Sciences, Bandar Abbas, Iran.
authors.forename: Azim
authors.initials: A
authors.lastname: Nejati Zadeh
authors.affiliation: Department of Bacteriology and Virology, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: Jamal
authors.initials: J
authors.lastname: Sarvari
keywords: Hepatic stellate cell; Liver fibrosis; SMAD4; shRNA