Brief Articles
Copyright ©2009 The WJG Press and Baishideng. All rights reserved.
World J Gastroenterol. Aug 14, 2009; 15(30): 3807-3813
Published online Aug 14, 2009. doi: 10.3748/wjg.15.3807
Connective tissue growth factor hammerhead ribozyme attenuates human hepatic stellate cell function
Run-Ping Gao, David R Brigstock
Run-Ping Gao, The Research Institute of Liver Diseases, First Hospital, Jilin University, Changchun 130021, Jilin Province, China
David R Brigstock, The Research Institute at Nationwide Children’s Hospital, Columbus, OH, United States; Division of Pediatric Surgery, Department of Surgery, The Ohio State University, Columbus, OH 43205, United States
Author contributions: Gao RP performed the experiments and wrote the manuscript; Brigstock DR planned and co-ordinated the study and edited the manuscript.
Correspondence to: Dr. David R Brigstock, Center for Cell and Developmental Biology, Research Institute at Nationwide Children’s Hospital, 700 Children’s Drive, Columbus, OH 43205, United States. david.brigstock@nationwidechildrens.org
Telephone: +1-614-3552824
Fax: +1-614-7225892
Received: April 29, 2009
Revised: June 18, 2009
Accepted: June 25, 2009
Published online: August 14, 2009
Abstract

AIM: To determine the effect of hammerhead ribozyme targeting connective tissue growth factor (CCN2) on human hepatic stellate cell (HSC) function.

METHODS: CCN2 hammerhead ribozyme cDNA plus two self-cleaving sequences were inserted into pTriEx2 to produce pTriCCN2-Rz. Each vector was individually transfected into cultured LX-2 human HSCs, which were then stimulated by addition of transforming growth factor (TGF)-β1 to the culture medium. Semi-quantitative RT-PCR was used to determine mRNA levels for CCN2 or collagen I, while protein levels of each molecule in cell lysates and conditioned medium were measured by ELISA. Cell-cycle progression of the transfected cells was assessed by flow cytometry.

RESULTS: In pTriEx2-transfected LX-2 cells, TGF-β1 treatment caused an increase in the mRNA level for CCN2 or collagen I, and an increase in produced and secreted CCN2 or extracellular collagen I protein levels. pTriCCN2-Rz-transfected LX-2 cells showed decreased basal CCN2 or collagen mRNA levels, as well as produced and secreted CCN2 or collagen I protein. Furthermore, the TGF-β1-induced increase in mRNA or protein for CCN2 or collagen I was inhibited partially in pTriCCN2-Rz-transfected LX-2 cells. Inhibition of CCN2 using hammerhead ribozyme cDNA resulted in fewer of the cells transitioning into S phase.

CONCLUSION: Endogenous CCN2 is a mediator of basal or TGF-β1-induced collagen I production in human HSCs and regulates entry of the cells into S phase.

Keywords: Connective tissue growth factor; Fibrosis; Hepatic stellate cell; Transforming growth factor-β1