title: [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].
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.
authors.affiliation: Department of Infectious Diseases, the Affiliated Hospital of Luzhou Medical College, Lu zhou Sichuan, China.
authors.forename: Yu-feng
authors.initials: YF
authors.lastname: Jiang
authors.forename: Hua-li
authors.initials: HL
authors.lastname: Sun
authors.forename: Jian-jun
authors.initials: JJ
authors.lastname: Zhang
authors.forename: Fei
authors.initials: F
authors.lastname: Huang
authors.forename: Jia-qun
authors.initials: JQ
authors.lastname: Liu
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D055513:Connective Tissue Growth Factor / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D005109:Extracellular Matrix / Q000378:metabolism*; D005786:Gene Expression Regulation; D020868:Gene Silencing; D055166:Hepatic Stellate Cells / Q000187:drug effects / Q000378:metabolism*; D006820:Hyaluronic Acid / Q000378:metabolism; D007797:Laminin / Q000378:metabolism; D008103:Liver Cirrhosis / Q000378:metabolism / Q000473:pathology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D051381:Rats; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism
chemical_list: C526316:CCN2 protein, rat; D024042:Collagen Type I; D007797:Laminin; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C576839:TIMP1 protein, rat; D019715:Tissue Inhibitor of Metalloproteinase-1; D016212:Transforming Growth Factor beta; D055513:Connective Tissue Growth Factor; D006820:Hyaluronic Acid