Open AccessSmall Interfering RNA‐mediated Caveolin‐1 Knockout on Plasminogen Activator Inhibitor‐1 Expression in Insulin‐stimulated Human Vascular Endothelial Cells
Author(s) -
YANG Huiling,
HE Shuya,
QUAN Zhihua,
PENG Weixia,
YAN Bin,
LIU Jianghua,
WEN Fang,
CAO Renxian,
XU Yangyan,
WEN Gebo,
HU Weixin
Publication year - 2007
Publication title -
acta biochimica et biophysica sinica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.771
H-Index - 57
eISSN - 1745-7270
pISSN - 1672-9145
DOI - 10.1111/j.1745-7270.2007.00270.x
Subject(s) - small hairpin rna , microbiology and biotechnology , rna interference , biology , plasminogen activator inhibitor 1 , gene silencing , reverse transcription polymerase chain reaction , small interfering rna , gene expression , gene knockdown , plasminogen activator , transfection , rna , cell culture , gene , biochemistry , genetics , endocrinology
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 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.