Repository of Research and Investigative Information

Repository of Research and Investigative Information

Hormozgan University of Medical Sciences

Blocking of SMAD4 expression by shRNA effectively inhibits fibrogenesis of human hepatic stellate cells

(2015) Blocking of SMAD4 expression by shRNA effectively inhibits fibrogenesis of human hepatic stellate cells. Gastroenterology and Hepatology from Bed to Bench.

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Abstract

Aim: 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. Patients and 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). Conclusion: 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. © 2015 RIGLD, Research Institute for Gastroenterology and Liver Diseases.

Item Type: Article
Additional Information: cited By 6
Keywords: alpha smooth muscle actin; messenger RNA; short hairpin RNA; Smad4 protein; tissue inhibitor of metalloproteinase 1; transforming growth factor beta, alpha SMA gene; Article; cell viability; COL 1A1 gene; controlled study; down regulation; fibrogenesis; gene silencing; gene targeting; genetic transfection; human; human cell; in vitro gene transfer; intracellular signaling; liver fibrosis; molecular pathology; protein expression; protein function; real time polymerase chain reaction; SMAD4 gene; stellate cell; TGF beta gene; TIMP 1 gene
Subjects: QU Biochemistry. Cell Biology and Genetics > QU 300-560 Cell Biology and Genetics
WI Digestive System > WI 700-770 Liver. Biliary Tract
Divisions: Research Vice-Chancellor Department > Infectious and Tropical Diseases Research Center
Depositing User: مهندس هدی فهیم پور
URI: http://eprints.hums.ac.ir/id/eprint/4368

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