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.
10.3760/cma.j.issn.1007-3418.2012.08.007
- VernacularTitle:结缔组织生长因子和基质金属蛋白酶组织抑制因子-1shRNA表达质粒对肝星状细胞细胞因子表达及细胞外基质分泌的影响
- Author:
Yu-feng JIANG
1
;
Hua-li SUN
;
Jian-jun ZHANG
;
Fei HUANG
;
Jia-qun LIU
Author Information
1. Department of Infectious Diseases, the Affiliated Hospital of Luzhou Medical College, Lu zhou Sichuan, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Cells, Cultured;
Collagen Type I;
genetics;
metabolism;
Connective Tissue Growth Factor;
genetics;
metabolism;
Down-Regulation;
Extracellular Matrix;
metabolism;
Gene Expression Regulation;
Gene Silencing;
Hepatic Stellate Cells;
drug effects;
metabolism;
Hyaluronic Acid;
metabolism;
Laminin;
metabolism;
Liver Cirrhosis;
metabolism;
pathology;
Polymerase Chain Reaction;
RNA, Messenger;
genetics;
metabolism;
RNA, Small Interfering;
genetics;
Rats;
Tissue Inhibitor of Metalloproteinase-1;
genetics;
metabolism;
Transfection;
Transforming Growth Factor beta;
metabolism
- From:
Chinese Journal of Hepatology
2012;20(8):576-580
- CountryChina
- Language:Chinese
-
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.
