title: Hepatic stellate cell-specific gene silencing induced by an artificial microRNA for antifibrosis in vitro.
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.
  
  
  OBJECTIVE
  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.
authors.affiliation: Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. changying@tjh.tjmu.edu.cn
authors.forename: Ying
authors.initials: Y
authors.lastname: Chang
authors.forename: Hua-jun
authors.initials: HJ
authors.lastname: Jiang
authors.forename: Xue-mei
authors.initials: XM
authors.lastname: Sun
authors.forename: Xiao-kun
authors.initials: XK
authors.lastname: Cai
authors.forename: Xing-xing
authors.initials: XX
authors.lastname: He
authors.forename: Pei-yuan
authors.initials: PY
authors.lastname: Li
authors.forename: Wang-xian
authors.initials: WX
authors.lastname: Tang
authors.forename: Yu-hu
authors.initials: YH
authors.lastname: Song
authors.forename: Ju-sheng
authors.initials: JS
authors.lastname: Lin
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics; D005240:Feasibility Studies; D020868:Gene Silencing; D005813:Genes, Synthetic; D055166:Hepatic Stellate Cells; D007438:Introns; D008103:Liver Cirrhosis / Q000235:genetics / Q000517:prevention & control*; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000235:genetics; D035683:MicroRNAs; D016133:Polymerase Chain Reaction; D051381:Rats; D017208:Rats, Wistar; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000235:genetics*
chemical_list: D024042:Collagen Type I; D035683:MicroRNAs; D019715:Tissue Inhibitor of Metalloproteinase-1; D053773:Transforming Growth Factor beta1; D020778:Matrix Metalloproteinase 2