Hepatic stellate cell-specific gene silencing induced by an artificial microRNA for antifibrosis in vitro.
Dig Dis Sci
; 55(3): 642-53, 2010 Mar.
Article
em En
| MEDLINE
| ID: mdl-19890714
ABSTRACT
BACKGROUND:
We previously reported that the anti-transforming growth factor-beta1 (TGF-beta1) ribozymes directed by T7 and CMV promoters could reverse the character of activated hepatic stellate cells (HSCs) in vitro and improve fibrotic pathology in vivo. However, nonspecific elimination of the effects of TGF-beta1 without selectivity might have unfavorable consequences, such as overwhelming inflammation, tissue necrosis, etc.AIMS:
To establish an activated-HSC-specific gene silencing method and validate its feasibility for antifibrosis in vitro.METHODS:
An artificial intronic microRNA (miRNA) expression system was established, containing three parts (1) a 1,074-bp SM-alpha actin promoter SMP8, which is a kind of RNA polymerase II promoter and has no activity in normal liver-derived cells but is switched on during the activation of HSCs, (2) intron1 modified by inserting an artificial pre-miRNA sequence against TGF-beta1, and (3) report gene enhanced green fluorescent proteins (EGFP). The feasibility of this system for artificial microRNA expression was validated through microRNA detection by real-time polymerase chain reaction (PCR). Alteration of biological characteristics of HSCs with the anti-TGF-beta1 miRNAs was preliminarily evaluated by measuring the expression levels of TGF-beta1 and its downstream molecules, including collagen I, matrix metalloproteinase 2 (MMP2), tissue inhibitor of metalloproteinase 1 (TIMP-1), etc.RESULTS:
The microRNA expression system could successfully produce mature anti-TGF-beta1 miRNAs in an activated-HSC-specific manner. The microRNA-induced inhibition rate of TGF-beta1 reached 70% and above. Accompanied by TGF-beta1 suppression, its downstream targets such as collagen I, MMP2, TIMP-1, etc. were also significantly downregulated in vitro.CONCLUSIONS:
Activated-HSC-cell-specific gene silencing could be induced well by the artificial intronic microRNA expression system to realize antifibrosis in vitro.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Inativação Gênica
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MicroRNAs
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Fator de Crescimento Transformador beta1
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Células Estreladas do Fígado
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Genes Sintéticos
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Cirrose Hepática
Limite:
Animals
Idioma:
En
Revista:
Dig Dis Sci
Ano de publicação:
2010
Tipo de documento:
Article
País de afiliação:
China