Connective tissue growth factor hammerhead ribozyme attenuates human hepatic stellate cell function

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. Semiquantitative RT-PCR was used to determine mRNA levels for CCN2 or collagen I, while protein levels of each molecule in cell /ysates 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.

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.