The Role Of MiR-451a In Regulating Angiotension Ⅱ-induced ICAM-1 Expression In Vascular Endothelial Cells And The Underlying Mechanism

Background:Vascular endothelial injury is the pathological basis for a variety of cardiovascular diseases.Abnormal expression of intercellular cell adhesion molecule-1(ICAM-1)in vascula vascular endothelial cells promotes adhesion and infiltration of monocytes/macrophages,leading to vascular endothelial dysfunction.Angiotensin Ⅱ(Ang Ⅱ),one of the major element hormones of the renin-angiotensin system,promotes the expression of ICAM-1.Micro RNAs(miRNAs)are a class of endogenous non-coding RNAs about 21-25 nt long found in eukaryotes that modulate gene expression at the post-transcriptional level,either by destabilizing m RNA or inhibiting translation.miR-451 a has been reported to be involved in the pathological processes of multiple diseases;however,the role of miR-451 a in the upregulation of Ang Ⅱ-induced ICAM-1 expresion in vascular endothelial cells remains unclear.Objective: To elucidate the role of miR-451 a in angiotensin Ⅱ-induced ICAM-1expression in vascular endothelial cells and the underlying molecular mechanism.Methods:1.Human umbilical vein endothelial cells(HUVECs)were treated with Ang Ⅱ(100nmol)for 6,12 and 24 hours.The expression of ICAM-1 and miR-451 a at m RNA and protein levels was detected by real-time quantitative PCR(q PCR)analysis.2.HUVECs were transfected with miR-451 a mimic,inhibitor or negative control(NC)for 24 hours and then treated with Ang Ⅱ(100 nmol)for 24 hours.The m RNA level of ICAM-1 was measured by q PCR analysis.The protein levels of ICAM-1,p-p38 MAPK,p38MAPK,p-p65 and p65 were detected by Western blot analysis.3.Target Scan 6.2 was applied to foercast the potential target gene(sphingosine-1-phosphate receptor 2,S1PR2)of expression of miR-451 a.Plasmids carrying S1PR23’-UTR wild type(WT)or mutants were constructed,and the dual luciferase gene reporter assay was performed in HEK-293 cells;The effect of miR-451 a mimic or miR-451 a inhibitor on protein level of S1PR2 in HUVECs was evaluated by Western blot analysis.4.Statistical analysis:All results are presented as mean±SEM.Graph Pad Prism 7 was used for statistical analysis and graphing.The student t test was used to compare the significant difference between two groups in normal distribution.The Mann-Whitney test was used if the data were not normally distributed.P values <0.05 were considered statistically significant.Results:1.ICAM-1 expression at both m RNA and protein levels were dramatically increased during 6-24 hours after Ang Ⅱ treatment in HUVECs,but miR-451 a expression was decreased at 12 and 24 hours after Ang Ⅱ treatment compared with saline control.2.Compared with miRNA blank control transfection of HUVECs with miR-451 a mimic remarkably increased miR-451 a expression,and dramatically inhibited the Ang Ⅱ-induced increase of ICAM-1,p-p38 MAPK and p-p65 protein levels.Conversely,transfection of miR-451 a inhibitor attenauted the expression level of miR-451 a and markedly enhanced Ang Ⅱ induced upregulation of ICAM-1,p-p38 MAPK and p-p65 protein levels.3.Target Scan 6.2 predicted the presence of miR-451 a conserved binding sequence in the 3’ untranslated region(3’-UTR)of S1PR2.Dual luciferase gene reporter assay revealed that miR-451 a mimic reduced S1PR2 luciferase activity.Western blot results confirmed that miR-451 a mimic remarkably inhibited Ang Ⅱ-induced upregulation of S1PR2 protein level in HUVECs,whereas miR-451 a inhibitor enhanceded Ang Ⅱ-induced increase of S1PR2 protein level.Conclusion: This study identifies that miRNA-451 a mediates ICAM-1 expression by regulating the target gene S1PR2 and its downstream mediators(p38 MAPK and NF-κB),and suggests that miR-451 a is a novel regulator for ICAM-1 expression in HUVECs.