title: Design and screening of antisense oligodeoxynucleotides against PAI-1 mRNA in endothelial cells in vitro.
abstract: OBJECTIVE
  To design and screen antisense oligodeoxynucleotides (ASODNs), which inhibit type-1 plasminogen activator inhibitor (PAI-1) expression in human umbilical vein endothelial cells (HUVEC) in vitro.
  
  
  METHODS
  Twenty seven ASODNs against different sites of PAI-1 mRNA were designed and transfected to HUVEC by lipofectin in vitro. The effects of ASODNs on PAI-1 antigen, PAI-1 activity and PAI-1 mRNA expression were detected by ELISA, amidolytical assay and RT-PCR, respectively.
  
  
  RESULTS
  Transforming growth factor beta1 (TGF-beta1)-treated HUVEC increased the expression of PAI-1 compared with the normal HUVEC. Five among twenty seven designed ASODNs were effective in inhibiting the increase in PAI-1 antigen and PAI-1 activity in a dose-dependent manner after 48-h transfection. In particular, ASODN 14 (AO14) exhibited the best inhibitory effect. The control sequences of AO14, including sense, scramble, and mismatch sequences, did not significantly inhibit PAI-1 activity. It was revealed that the inhibitory efficacy of AO14 was in a sequence-specific manner. RT-PCR showed that ASODN 1, 7, 8, 14, and 15 decreased PAI-1 mRNA expression induced by TGF-beta1 and AO14 showed the best inhibitory effect.
  
  
  CONCLUSIONS
  ASODN 1, 7, 8, 14, and 15, among twenty seven designed ASODNs against PAI-1 mRNA, significantly decreased PAI-1 antigen and PAI-1 activity induced by TGF-beta1 in a dose-dependent manner in HUVEC in vitro. AO14 showed the best inhibitory effect on PAI-1 expression in a sequence-specific manner. The results of RT-PCR indicated that inhibitory effects of ASODNs on PAI-1 biosynthesis occurred at the mRNA level. Four among five effective target sites of ASODNs located at the translation initiation site or within the translation area of PAI-1 mRNA, suggesting that these sites may be promising sites for the design of effective ASODNs.
authors.affiliation: Beijing Institute of Radiation Medicine, Beijing 100850, China.
authors.forename: Qi-Sheng
authors.initials: QS
authors.lastname: Jiang
authors.forename: Sheng-Qi
authors.initials: SQ
authors.lastname: Wang
mesh_terms: D001483:Base Sequence; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D020319:Oligodeoxyribonucleotides, Antisense / Q000008:administration & dosage / Q000138:chemical synthesis* / Q000235:genetics / Q000494:pharmacology*; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology; D014471:Umbilical Veins / Q000166:cytology
chemical_list: D020319:Oligodeoxyribonucleotides, Antisense; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D053773:Transforming Growth Factor beta1