The Regulation Effect And Mechanism Research Of MiR-19b On TNF-? Induced Vascular Endothelium Injury

Background:Atherosclerosis is the common pathologic basis of cardiovascular diseases such as myocardial infarction and the main cause of death.Tumor necrosis factor-?(TNF-?),an important inflammatory factor,induces endothelial cell injury,which results in endothelial dysfunction,initiating the atherosclerosis progress.Recently,lots of miRNA have been proved playing important role in regulating endothelial cell function,enhancing angiogenesis and endothelium.Researchers have noted that the miR-17-92 cluster is significantly downregulated among patients with atherosclerotic diseases.This indicates that the miR-17-92 cluster may be involved in the functions of vascular endothelium of patients with atherosclerosis.However,the mechanism of the miR-17-92 cluster in repairing endothelial cel injury remains unclear.Objective:In this research,we investigate the effect and mechanism of the miR-17-92 cluster on TNF-? induced endothelial cell dysfunction.We hope that the results of our search could help to find new ideas and methods for atherosclerotic diseases diagnosis and treatment.Methods and Results:(1)Twelve patients diagnosed with coronary arteary disease(CAD)by our Division ofCardiology and twelve healthy donors were recruited.A 10 ml sample of peripheral blood was collected in an EDTA-containing vacutainer tube from eachindividual to detect the miR-17-92 cluster expression by qRT-PCR.Among the miR-17-92 family of miRNAs,the levels of miR-19 b and miR-92 a were significantlylower in patients with CAD than in healthy subjects,.However,nodifferences were observed with respect to the levels of miR-17?miR-18a?miR-19 a and miR-20 a between the two groups.(2)Isolation and identification of HUVECs.Primary human umbilical vein endothelial cells(HUVECs)were isolated and cultured following a previously described protocol.Through the morphological observation and the factors(CD31 and vWf)in cell surface identification,endothelial cel s passagesfrom three to five were used for this study.(3)Apoptosis of HUVECs is induced by TNF-?.Apoptosis of endothelial cells treated with 0,1,10,50 or 100 ng/ml of TNF-? for 24 h was measured by flow cytometry and TUNEL/DAPI-stained cell photomicrography.Both results showed that TNF-? induces endothelial cell apoptosis in a dose-dependent manner and that the lowest effective dose of 24 h TNF-? treatment was 10 ng/ml.(4)We used a microarray chip to analyze changes in miRNA expression between 3 TNF-?treated groups and 3 control groups.The results in TNF-?treated groups revealed that 18 miRNAs(6 of which were upregulatedand 12 of which were downregulated)exhibited a significant change compared with the control group.The expression of miR-19 b among the miR-17-92 cluster was significantly decreased following 24 h of TNF-? treatment.(5)Changes in miRNA-19 b levels of the HUVECs at 24 h following transfection with the vehicle,the miR-19 b mimic,the mimic control,the miR-19 b inhibitor or the inhibitor control were detected by qRT-PCR.The miR-19 b mimic upregulated,whereas the miR-19 b inhibitor downregulated miR-19 b expression.Flow cytometry and TUNEL/DAPI-stained cell photomicrography were used to detect cell apoptosis levels.In comparison with the control group,the miR-19 b mimic could significantly decrease endothelial cell apoptosis,whereas the miR-19 b inhibitor increase endothelial cell apoptosis.(6)According to several publicly available bioinformaticsweb sites(TargetScan,miRanda and miRBase),both Apaf-1,PTEN and Casp7 may be direct targets ofmiR-19 b.The luciferase reporter plasmids and function experiments further demonstrated that Apaf-1,PTEN and Casp7 were direct target genes of miR-19 b.(7)In TNF-? induced endothelial cell apoptosis model,the miR-19 b inhibitors and Apaf1-siRNA or PTEN-siRNA were cotransfected into HUVECs in order to test the role that miR-19 b,Apaf-1 and PTEN may play in.The results showed that miR-19 b could attenuate TNF-? induced endothelial cell dysfunction via the Apaf1/Caspase pathway and the PTEN/Caspase pathway.Conclusion:In conclusion,the downregulated expression levels of miR-19 b in the CAD patients may be one factor which causes endothelial cell dysfunction.miR-19 b could attenuate TNF-? induced endothelial cell injury via the Apaf1/Caspase pathway and the PTEN/Caspase pathway.