Small interfering RNA-mediated caveolin-1 knockout on plasminogen activator inhibitor-1 expression in insulin-stimulated human vascular endothelial cells.
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 (1x10(-9) to 1x10(-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 CAV-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.
Yang
Huiling
H
Institute of Clinic Medicine, the First Affiliated Hospital of Nanhua University, Hengyang, China.
He
Shuya
S
Quan
Zhihua
Z
Peng
Weixia
W
Yan
Bin
B
Liu
Jianghua
J
Wen
Fang
F
Cao
Renxian
R
Xu
Yangyan
Y
Wen
Gebo
G
Hu
Weixin
W
D001483:Base Sequence; D051242:Caveolin 1 / Q000037:antagonists & inhibitors / Q000235:genetics*; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*
C495540:CAV1 protein, human; D051242:Caveolin 1; D017931:DNA Primers; D007328:Insulin; D017395:Plasminogen Activator Inhibitor 1; D034741:RNA, Small Interfering