title: Connective tissue growth factor hammerhead ribozyme attenuates human hepatic stellate cell function.
abstract: OBJECTIVE
  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)-beta1 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-beta1 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-beta1-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.
  
  
  CONCLUSIONS
  Endogenous CCN2 is a mediator of basal or TGF-beta1-induced collagen I production in human HSCs and regulates entry of the cells into S phase.
authors: Research Institute of Liver Diseases, First Hospital, Jilin University, Changchun 130021, Jilin Province, China.
authors: Run-Ping
authors: RP
authors: Gao
authors: David R
authors: DR
authors: Brigstock
mesh_terms: D002453:Cell Cycle / Q000502:physiology; D002460:Cell Line; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D055513:Connective Tissue Growth Factor / Q000235:genetics / Q000378:metabolism*; D055166:Hepatic Stellate Cells / Q000502:physiology*; D006801:Humans; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism
chemical_list: C526317:CCN2 protein, human; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D016337:RNA, Catalytic; D053773:Transforming Growth Factor beta1; C427204:hammerhead ribozyme; D055513:Connective Tissue Growth Factor