Roles And Discussions On Mechanism Of MiR-4463 In Vascular Endothelial Cells Apoptosis Induced By High Glucose

Objective:Long-term hyperglycemia in patients with diabetes can damage endothelial cells through multiple pathways,which plays an important role in the development of diabetic macrovascular complications.The aim of this study was to investigate the expression of miR-4463 in diabetes patients plasma,and further explore the roles and mechanism of miR-4463 in human umbilical vein endothelial cell（HUVECs）apoptosis induced by high glucose.It is hoped to provide a new target for the analysis of vascular endothelial cell injury mechanism in diabetic condition,and to seek new ideas for the prevention and treatment of diabetic macrovascular complications.Methods:（1）The plasma of 20 controls and 20 diabetic patients were extracted,and the expression level of miR-4463 was detected by Real time quantitative PCR.（2）HUVECs were identified by detecting the expression of FⅧand CD31 via immunefluorescence staining.（3）After25 mmol/L glucose treatment of HUVECs for 24 h,48 h and 72 h,the expression level of miR-4463 was detected by RT-q PCR.Then selected the most significant time points for follow-up experiments.（4）miR-4463mimics,miR-4463 inhibitor and Negative control transfected HUVECs to up or down regulation the levels of miR-4463 for 24 h,then high glucose treated for 24 h:（1）The cell vitality of HUVECs was detected by CCK-8.（2）The apoptosis of HUVECs was detected by flow cytometry.（3）The expression of apoptosis-related proteins XIAP,Bcl-2,Bax and cleaved caspase 3 were detected by western blot.（5）Targetscan and miRDB analysis were used to predict the potential target genes of hsa-miR-4463.Up-regulating or down-regulating the expression of miR-4463,then detected the changes of PNUTS m RNA by PCR,and the expression of PNUTS protein by western blot.（6）Detecting the changes of AKT signaling pathway related proteins such as cleaved caspase 9,p-AKT Ser473,total AKT,p-Bad Ser136 and total Bad by western blot after down-regulating the expression of miR-4463.Results:（1）The expression of miR-4463 in the plasma of diabetic patients was about 15.77 times than that of control group（P<0.05）.（2）TheⅧfactor related antigen（red fluorescence）expressioned in the cell cytoplasm,and the CD31（red fluorescence）expressioned in the cell membrane or cell connections,which confirming the HUVECs were in good endothelial cells characteristic.（3）The expression level of miR-4463was increased significantly（P<0.05）in HUVECs after high glucose treatment for 24h,48h,72h,and it was 2.96±0.31、2.41±0.17、2.52±0.23times than that of control group.But there was no statistically significant difference between 24h,48h,72h groups（P>0.05）.The expression level of miR-4463 increased the most obviously in 24h group compared with control group.（4）The absorbance of the NC group,the over-expression group and the inhibiting group was 0.56±0.08,0.51±0.09 and 0.68±0.08,and the absorbance of the inhibiting group was significantly higher than that of the NC group（P<0.05）.The absorbance of over-expression group was slightly lower than that of NC group,and there was no statistically difference（P>0.05）.（5）（1）The apoptosis rate of the normal control group was 3.4%±0.3%,and the high-glucose group was 26.7%±3.6%,suggesting that the apoptosis rate was significantly increased with high glucose（P<0.05）.（2）the apoptosis rate of NC group,the over-expression group and the inhibiting group was 28.2%±3.4%,30.1%±30.1%and15.4%±2.6%,suggesting there was no statistically significant difference between the over-express group and the NC group（P>0.05）,while the apoptosis rate of inhibiting group was significantly decreased（P<0.05）compared with NC group.（6）The gray value of the NC group was set to 1.Compared with the NC group,the expression of XIAP（thegray value was 1.89±0.18）and Bcl-2（1.30±0.19）was increased,while the expression of cleaved casepase 3（0.45±0.06）and Bax（0.33±0.07）was inhibited,and the proportion of Bcl-2/Bax was significantly increased（P<0.05）.（7）（1）There was no significant difference of PNUTS m RNA level between the over-expression group(2^-ΔΔCt=0.86±0.21)and inhibiting group(2^-ΔΔCt=1.12±0.24)compared with NC group（P>0.05）.（2）While the over-expression group significantly inhibited the expression of PNUTS protein（0.48±0.18,P<0.05）,and the inhibiting group could increase the expression of PNUTS protein（1.97±0.21,P<0.01）.（8）Compared with NC group,（1）the level of PNUTS was increased（1.90±0.21,P<0.05）.（2）the expression of cleaved caspase 9 was restrained（0.64±0.10,P<0.05）,（3）the ratio of p-AKT/AKT and p-Bad/Bad was increased（P<0.05）in inhibiting group.Conclusion:（1）The expression level of miR-4463 in patients with T2DM or in HUVECs treated with high-glucose is significantly increased compared with the control group,suggesting that miR-4463 may be related to the development of diabetes.（2）Down-regulating of miR-4463could inhibit the apoptosis of HUVECs induced by high glucose.（3）PNUTS may be a potential target genes of miR-4463,and miR-4463regulates the expression of PNUTS after transcription.（4）miR-4463 is involved in regulation of the vascular endothelial cell apoptosis induced by high-glucose by regulating the expression of PNUTS and affecting the AKT signaling pathway.