MicroRNAs-21’Functional Study And The Expression Differences Of It In Vascular Endothelial Cell By High Glucose Environment And In Coronary Heart Disease Patients

Objective:This project intends to use the vascular endothelial cells in vitro models and clinical samples, to study the expression of differences of miRNA-21in endothelial cells in high glucose environment.Cellular and molecular biology method was used to these biological functions of miRNA-21, and then preliminary expound the molecular mechanisms of miRNA-21in the regulation of endothelial cell physiological activities in high glucose condition.Methods:All hospitalized patients who received the selective coronary angiography though the Seldinger puncture method in the femoral artery, into6F/7F sheath, to discard get the blood sample of approximately2ml at the sheath end, and then extract10ml blood sample to anticoagulant ltube, using immunomagnetic beads (containing beads direct subject of anti-CD34antibody) separation of free endothelial cells in the blood samples were frozen at-80℃; treated by with a concentration of30mM glucose solution in human umbilical vein endothelial cells (HUVECs)24hours before put-80℃frozen.The miRNA-21expressions of cell was detected by RT-PCR method. miRNA-21inhibitor2’OMe-miRNA-21transfected by LipofectamineTM2000to HUVECs incubated by DMEM.HUVECs were treated high glucose for48hours,collecting cells for two-dimensional electrophoresis analysis and identify differentially expressed proteins by mass spectrometry,then verify the differences in protein expression levels by Western-blot analysis.Results:1, compared with the CAD group and control group, the proportion with a history of smoking,age, weight, homocysteine, and microRNA21were significantly higher than control group, There were statistically differences between them (P<0.05).2, Among the four groups, age, smoking history, homocysteine. miR-21were statistically differences (P<0.05).3, high-sugar significantly up-regulated the expression of miR-21in vitro cultured human umbilical vein endothelial cells.(P<0.05).4, There are31differentially expressed proteins were identified in two-dimensional electrophoresis of HUVECs. in anti-miR-21group,13up-regulated,18down-regulated,in which,4protein have the most significant differences, annexin A2(ANNEXIN A2) S100A4、mitochondrial superoxide dismutase (the SOD2),and thioredoxin (thioredoxin) was respective identified by mass spectrometry.5、The expression of ANNEXIN A2S100A4, the SOD2, Thioredoxin were all down-regulated in the anti-miR-21group By western-blot analysis of differentially expressed proteinConclusion:1、The expression levels of miRNA-21of vein endothelial cell in Patients with coronary heart disease is significantly higher than those without CHD.2、high-sugar significantly up-regulated the expression of miR-21in vein endothelial cells3、increasing the expression levels of miRNA-21up-regulated the expressions of ANNEXIN A2,S100A4,the SOD2,Thioredoxin...