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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 collagenⅠ,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 collagenⅠ,and an increase in produced and secreted CCN2 or extracellular collagenⅠprotein levels.pTriCCN2-Rz-transfected LX-2 cells showed decreased basal CCN2 or collagen mRNA levels,as well as produced and secreted CCN2 or collagenⅠprotein. Furthermore,the TGF-β1-induced increase in mRNA or protein for CCN2 or collagenⅠwas 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 collagenⅠproduction in human HSCs and regulates entry of the cells into S phase.
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 collagenⅠprotein. Furthermore, the TGF-β1-induced increase in mRNA or protein for CCN2 or collagenⅠwas partially inserted into 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 production in human HSCs and regulates entry of the cells into S phase.