Design and screening of antisense oligodeoxynucleotides against PAI-1 mRNA in endothelial cells in vitro


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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.


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.


Transforming growth factor β1(TGF-β1)-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-β1 and AO14 showed the best inhibitory effect.


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-β1 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.


Project supported by the National Natural Science Foundation of China (No 30271479) and National Hi-TECH research and development program of China (No 2001AA234041) and Post-doctor Science


Foundation of China (No 2001-29).


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