The 27nt-miRNA Suppresses ENOS Expression And Endothelial Cell Proliferation By A Mechanism Related To The Transcription Factor AP-1

Objective:We previously reported that endothelial nitric oxide synthase intron 4 of 27 bp repeat promoter to produce the corresponding 27nt miRNA, which regulated expression of eNOS.This study was to investigate an intronic miRNA regulated expression of the human endothelial nitric oxide synthase gene and proliferation of endothelial cells by a mechanism related to the transcription factor AP-1。1. The functional role of 27nt miRNA involved in HUVECs phenotype was studied.2. The mechanisms 27nt miRNA involved in regulating HUVECs proliferation and metastasis.3. To explore the regulatory role and the underlying mechanisms 27nt miRNA in HUVECs invasion and migration, with the aim of better elucidating the mechanisms of HUVECs metastasis and laying a foundation for formulating novel therapeutic target for treatment of HUVECs.Methods:1..Observe the effects of 27nt miRNA on biological behavior of HUVECs in vitro:HUVECs were transfected with wt-27nt miRNA å'Œ mut-27nt miRNA mimics respectively, then MTT assay and clone were used for evaluating cell proliferation, Migration ability of endothelial cells was measured by scratch wound model in vitro.,Transwell migration and invasion assays were employed for investigating the metastatic properties.2. Prediction and validation of 27nt miRNA targets:By using miRanda and,PicTar.We speculated that AP-1 might be one of the target genes of 27nt miRNA in HUVECs. Luciferase reporter assay was used to verify 27nt miRNA directly regulated AP-1 expression. Expression of eNOS and activator protein 1 (AP-1) was measured by real-time RT-PCR (mRNA level) and Western Blotting (protein level),3. Under the stimulus VEGF growth factor,expression of eNOS and activator protein 1 (AP-1) was measured by real-time RT-PCR (mRNA level) and Western Blotting (protein level),Results:1. Overexpression of 27nt-miRNA significantly inhibited endothelial cells proliferation, invasion and migration in vitro.2. Bioinformatics predicted that AP-1 might be a target of 27nt-miRNA Luciferase reporter assay showed that 27nt-miRNA over-expression remarkably repressed the expression of luciferase from a vector also containing the wild-type 27nt-miRNA binding site (AP-1-UTR), but had no effect on mutant and negative control vector. We previous work found that eNOS is not the target.And, the eNOS and AP-1 expression at mRNA and protein levels were down-regulated by overexpression of 27nt-miRNA. These data demonstrate 27nt-miRNA is involved in HUVECs invasion and metastasis through negatively regulating AP-1 expression.3. Under the stimulus VEGF growth factor, expression of eNOS and activator protein 1 (AP-1)(mRNA level) and Western Blotting (protein level) was decreased compare to control,Conclusions:1. These data demonstrate that overexpression of 27nt-miRNA inhibits endothelial cell proliferation, invasion and migration.2. Moreover, overexpression of 27nt-miRNA suppresses AP-1 expression and eNOS gene expression in endothelial cells. Therefore, overexpression of 27nt-miRNA inhibits endothelial cell proliferation, invasion and migration.3. Under the stimulus VEGF growth factor, expression of eNOS and activator protein 1 (AP-1) (mRNA level) and Western Blotting (protein level) was decreased compare to control,...