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Effect of shRNA-mediated silencing of CTGF and TIMP-1 on mRNA expression of CTGF, TIMP-1, and PC I and secretion of extracellular matrix in rat hepatic stellate cells / 中华肝脏病杂志
Chinese Journal of Hepatology ; (12): 576-580, 2012.
Article in Chinese | WPRIM | ID: wpr-296846
ABSTRACT
To investigate the effect of short hairpin RNA (shRNA)-mediated silencing of CTGF and TIMP-1 in hepatic stellate cells (HSCs) on mRNA expression of TIMP-1, CTGF, and procollagen type-I (PC I), as well as secretion of extracellular matrix (ECM) proteins. Two recombinant expression plasmids harboring shRNAs against CTGF and TIMP-1 (psiRNA-GFP-CTGF and psiRNA-GFP-TIMP-1) were transfected alone or together into TGFb1-activated HSC-T6 cells. The mRNA expression levels of CTGF, TIMP-1, and PC I were detected by fluorescence quantitative PCR (FQ-PCR). The concentrations of secreted PC type-III, hyaluronate (HA), and laminin (LN) were measured by radioimmunoassay (RIA) of culture supernatants. FQ-PCR analysis showed that CTGFshRNA and TIMP-1shRNA specifically inhibited the expression of CTGF, TIMP-1, and PC I mRNA in activated HSC-T6 cells. The concentrations of secreted PC III, HA, and LN were decreased significantly in HSC-T6 cells with shRNA-silenced CTGF or TIMP-1 (P less than 0.01 or P less than 0.05). Moreover, HSC-T6 cells with shRNA-silenced CTGF and TIMP-1 showed a more robust decrease in synthesis of PC III, HA and LN (all, P less than 0.01), as well as in mRNA expression of PC I (P less than 0.05). CTGFshRNA and TIMP-1shRNA effectively inhibit expression of the respective target genes, as well as of PC I, and decrease secretion of ECM components from HSC-T6 cells. Silencing of both CTGF and TIMP-1 produces more robust effects than either in isolation. These data suggest that CTGF and TIMP-1 may be effective targets of shRNA-based gene therapy to treat liver fibrosis.
Subject(s)
Full text: Available Index: WPRIM (Western Pacific) Main subject: Pathology / RNA, Messenger / Transfection / Down-Regulation / Cells, Cultured / Gene Expression Regulation / Polymerase Chain Reaction / Transforming Growth Factor beta / Laminin / Tissue Inhibitor of Metalloproteinase-1 Limits: Animals Language: Chinese Journal: Chinese Journal of Hepatology Year: 2012 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Main subject: Pathology / RNA, Messenger / Transfection / Down-Regulation / Cells, Cultured / Gene Expression Regulation / Polymerase Chain Reaction / Transforming Growth Factor beta / Laminin / Tissue Inhibitor of Metalloproteinase-1 Limits: Animals Language: Chinese Journal: Chinese Journal of Hepatology Year: 2012 Type: Article