The Role Of Insulin In The Expression Of HMGA1in Human Umbilical Vein Endothelial Cells

Objective: To explore the effect of insulin on the expression of HMGA1in humanumbilical vein endothelial cells and to unravel the mechanism by which the HMGA1is induced,providing a further explanation for the association between insulin andpathogenesis of diabetic atherosclerosis.Methods: Human umbilical vein endothelial cells HUVEC-12cell lines cultured invitro were utilized in the present study to investegate the role of insulin in theexpression of HMGA1. Our experiments were divided into four parts. The first part isto inveatigate the effect of insulin on HMGA1expression. The endothelial cells weretreated with different concentrations of insulin (10^-11mol/L,10^-10mol/L,10^-9mol/Lgroup,10^-8mol/L and10^-7mol/L) for24hours. HMGA1expression was measuredby semiquantitatve reverse transcription PCR and immunofluorescence staining.Insulin (10^-9mol/L) were added to culture endothelial cells for2hours,4hours,8hours,16hours and24hours and HMGA1expression was detected by RT-PCR. Thesecond part is to investegate the role of PI3K signalling in the effect of insulin onHMGA1expression. The endothelial cells were pre-treated with different kinds andconcentrations of the PI3K pathway inhibitor (including the control group,10^-9mol/Linsulin group,50μmol/L and100μmol/L of wortmanin plus insulin group,10μmol/L and20μmol/L of LY294002plus insulin group) for2h, and cells were mantainedin medium with10^-9mol/L of insulin. The role of PI3K signaling pathway ininsulin induced HMGA1expression was observed in endothelial cell by RT-PCR andimmunofluorescence. The third part is to unveil the effect of insulin on HMGA1promoter activity. PGL4.10plasmid containing HMGA1promoter was transfectedinto endothelial cell, and then different concentration of insulin was added toendothelial cells and cultured for another8hours. The promoter activity of HMGA1was detected by the luciferase assay. Meanwhile, PI3K inhibitors effect on insulin regulating HMGA1promoter activity was detected with the same methods as above.The last part investigated the role of SP1in insulin (control group,10^-11mol/L group,10^-9mol/L group,10^-7mol/L group) induced HMGA1expression by the use ofChIP-PCR technique.Results:1)The effect of insulin on HMGA1expression. HUVEC-12were treatedwith different concentrations of insulin for24h respectively. Compared with controlgroup, the level of HMGA1mRNA was increased at the concentration of10^-9mol/Linsulin and10^-8mol/L insulin（p<0.05）.HMGA1protein expression level was foundto be higher than those in other groups at the concentration of10^-9mol/L insulin（p<0.05）. When the cells were treated with10^-9mol/L insulin for different time,HMGA1mRNA level was significant upregulated at8hours and16hours（p<0.05）and reached the peak at8hours.2) Role of PI3K signalling in insulin-inducedHMGA1expression. The endothelial cells were treated with two differentconcentration of the PI3K pathway inhibitor（Wortmanin and LY294002） and thentreated with10^-9mol/L insulin.Wortmanin could not block the insulin inducedHMGA1mRNA（p>0.05）, while insulin induced HMGA1mRNA were significantinhibited by LY294002（p<0.05）in HUVEC-12.3) The effect of insulin on HMGA1promoter activity. PGL4.10plasmid with HMGA1promoter was transfected intoendothelial cell, and then different concentration of insulin was added to endothelialcells for24h and luciferase reporter assay sgowed that HMGA1promoter activitywere upregulated in all insulin treated group compared to the control group（p<0.05）.Then endothelial cells transfected with HMGA1promotor were treated with10^-9mol/L insulin for0h，2h,4h,8h and it was showed that HMGA1promoter activity wereupregulated by the the treatment of insulin for2hours,4hours and8hours（p<0.05）,reaching the peak at8hours. The endothelial cells transfected with HMGA1promotorwere treated with two different concentrations of the PI3K pathway inhibitor. It wasfound that Wortmanin could not block on the promoter activity of HMGA1inducedby insulin （p>0.05）, and the insulin induced promoter activity of HMGA1weresignificant inhibited by LY294002（p<0.05）.4) The role of SP1in insulin induced HMGA1expression. ChIP PCR showed that the binding affinity of SP1with HMGA1promoter was increased by10^-9mol/L insulin in endothelial cells, which indicatesthat insulin might induce HMGA1expression by increasing the binding ability of SP1to HMGA1promotor.Conclusion:1)Insulin could upregulate HMGA1expression in endothelial cellsthrough phosphatidylinositol3-kinase （PI-3K） pathway.2)Insulin could increase thebinding affinity of SP1to HMGA1promoter in endothelial cells.