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Using human vascular endothelial cells (ECV304) as the target, we studied the effect of caveolin (CAV)-1 in the course of insulin-stimulated expression of plasminogen activator inhibitor (PAI)-1. The appropriate single-stranded oligonucleotides representing the RNAi CAV-1 gene were analyzed by Ambion software. After annealing to generate double-stranded oligonucleotides (ds oligo), it was cloned into the pENTR/U6 entry vector containing RNA polymerase III expression element by T4 DNA ligase. The short hairpin (shRNA) sequences transferred from the pENTR/U6 entry were cloned into the pLenti6/BLOCK-iT-DEST vector with an LR recombination reaction. After identification by sequencing, we successfully constructed the CAV- 1 RNAi lentiviral expression system using Gateway technology. Silencing efficiency was assayed by real-time reverse transcription-polymerase chain reaction, immunofluorescence staining and Western blotting. ECV304 cells were cultured in the medium containing different concentrations of insulin (1×10−9 to 1×10−7 M) with the CAV-1 gene silenced or not. The expression level and subcellular localization of PAI-1 and CAV-1 were compared using reverse transcription-polymerase chain reaction, immunofluorescence staining and Western blot assay. The results showed that the potent inhibition of C AV- 1 expression could reach 85%, and it was specific to the CAV-1-derived shRNA, not the S100A13-derived shRNA. There was no dramatic difference in PAI-1 expression between the RNAi+ and RNAi- ECV304 cells incubated with physiological insulin, but PAI-1 protein did accumulate under the cell membrane. As the concentration of insulin increased, the expression of PAI-1 was up-regulated, whereas the expression of CAV-1 attenuated. Furthermore, PAI-1 clearly augmented after CAV- 1 knockdown. These results indicated that hyperinsulinism could promote PAI-1 expression by inhibiting CAV-1, and stabilizing or up-regulating CAV- 1 expression in endothelial cells might reduce complications of the great vessels and capillary vessels in diabetes.