VEGFA Modulated By PBMC-Derived MiR-4306 Determines Human Coronary Artery Endothelial Cell Migration And Proliferation

Background: Mcroparticles derived from human peripheral blood mononuclear cells involved in many pathophysiologic pathways of coronary heart disease,such as lipid metabolism disorders,inflammatory immune responses.Our previous study found that the expression of miR-4306 in MP derived from PBMC in patients with coronary heart disease was significantly increased which compared with control group.However,the diseases relationship between miR-4306 in MP derived from PBMC and coronary heart disease and the mechanism of miR-4306 on human coronary artery endothelial cells are still unclear.Objective: The purpose of this study was to investigate diseases relationship between miR-4306 in MP derived from PBMC and coronary heart disease and the mechanism of miR-4306 on HCAECs.Methods:1.Clinical trial group: Blood samples were obtained from control group(healthy people)and patients with coronary heart disease.The groups were as follows: control group,single vessel lesion group,multivessel disease group;control group,elective PCI group and emergency PCI group.2.Cell experiment grouping: According to different experimental purposes,the groups were as follows: PBMC-MP control group,PBMC-MP elective PCI group,PBMC-MP emergency PCI group;Control group,miR-4306 mimics group;Control group,miR-4306 inhibitor group.3.Experimental objects were taken 9 ml whole blood and extracted PBMC-MP.Whether MP derived from PBMC was confirmed by flow cytometry.The expression level of miR-4306 in PBMC-MP which derived from healthy people and patients with coronary heart disease was detected by qRT-PCR.4.Whether miR-4306 can regulate the expression of SLC16A2 gene and VEGFA gene was detected by dual luciferase reporter gene assay.5.Whether HCAECs swallowed MP was detected by immunofluorescence and the expression level of miR-4306 was detected at different time by qRT-PCR after MP was devoured by HCAECs.After MP was devoured by HCAECs,the protein expression level of SLC16A2 and VEGFA and the ability of lactic acid metabolism,cell migration and proliferation were detected respectively by western blot,Lactic acid kit,Transwell cell and CCK8.6.HCAECs was transfected respectively with Negative control ?Hsa-micro-4306-mimics?MicroRNA inhibitor N.C?Hsa-micro-4306-inhibitor.Then the expression level of miR-4306,the protein expression level of SLC16A2 and VEGFA,the ability of lactic acid metabolism,cell migration and proliferation were also detected respectively by qRT-PCR,western blot,Lactic acid kit,Transwell cell and CCK8.Results:1.Compared with control group,the expression of miR-4306 in PBMC-MP of single vessel lesion group and multiple vessel lesion group were significantly increased(P<0.05 and P<0.01),and the expression of miR-4306 in multiple vessel disease group was significantly higher than that in single vessel disease group(P<0.05).Compared with control group,theexpression of miR-4306 in PBMC-MP of elective PCI group and emergency PCI group were increased(P<0.05 and P<0.01),and the expression of miR-4306 in emergency PCI group was also significantly higher than that in elective PCI group(P<0.01).2.The results of dual luciferase reporter gene assay showed that Hsa-miR-4306 had moderate target regulating to SLC16A2 gene and VEGFA gene.3.The phagocytosis of MP by HCAECs was the strongest at 12-24 h and24-36 h.The expression of miR-4306 increased abruptly at 12 h,reached the highest peak at 36 h and then began to decline after HCAECs swallowed MP.4.There was no significant difference in the protein expression of SLC16A2 in PBMC-MP control group,PBMC-MP elective PCI group,PBMC-MP emergency PCI group(P>0.05).And there was no significant change in HCAECs lactate metabolism(The value of lactic acid metabolism was 11.47�0.72 mM,13.4�0.53 mM,14.2�0.39 mM,P>0.05).The protein expression of VEGFA in PBMC-MP elective PCI group was significantly lower than that in PBMC-MP control group(P<0.05).Compared with PBMC-MP control group,the migration ability of HCAECs in PBMC-MP elective PCI group was significantly decreased(P<0.05)and the proliferative ability of HCAECs was significantly decreased at 24-48 h(P<0.05).5.Compared with control group,miR-4306 mimics group and miR-4306 inhibitor group significantly increased and decreased the expression of miR-4306 in HCAECs(P<0.01 and P<0.01),as well as inhibited and up-regulate the protein expression of SLC16A2 and VEGFA(P<0.01 and P<0.01;P<0.05 and P<0.05).Compared with control group,the capability of lactic acid metabolism had no difference in miR-4306 mimics and miR-4306 inhibitor group(The value of lactic acid metabolism was 12.86�0.29 mM,11.43�0.41 mM,12.14�0.18 mM,13.57�0.57 mM,P>0.05).Compared with control group,miR-4306 mimics group and miR-4306 inhibitor group respectively decreased and increased the migration ability of HCAECs(P<0.01 and P<0.01),but did not change the proliferation ability of HCAECs(P>0.05 and P>0.05).Conclusion:1.miR-4306 in CHD patients inhibited the target protein expression of VEGFA,reducing the migration and proliferation ability of HCAECs.Although miR-4306 inhibited the target protein expression of SLC16A2,there was no effect on the lactate metabolism ability of HCAECs.2.Compared with control group,the expression of miR-4306 in PBMC-MP increased in patients with coronary heart disease,and it was positively correlated with the severity of coronary artery disease and the severity of the disease.