| Objective:To construct recombinant adenoviruses of microRNA-145（mi RNA-145）overexpression and interference,and to investigate their effects on proliferation and angiogenesis of HUVECs and its underlying mechanism.Methods:The shuttle plasmid of pHBAd-EF1-MCS-GFP and pHBAd-U6-CMV-GFP were used to construct miR-145 overexpression and interference adenoviruses,motifs of which were determined by gene sequencing.After packaged with backbone plasmids of p HBAd-BHG and amplified in HEK293 cells,the infection titer of virus was detected by TCID500 assay.MiRNA-145 level was detected by qRT-PCR.Cell proliferation was evaluated by MTT,CCK8 and Western bloting for PCNA.Angiogenesis was assessed by Tube formation assay.Target genes and interaction between miR-145 and cAMP responsive element binding protein（CREB）were predicted by bioinformatics softwares,miRanda and mirWalk.Then CREB and VEGF proteins after virus transfection were determined by Western blot.And the binding site of miR-145 to CREB3’UTR was examined by Luciferase reporter assay.Results:The motifs of miRNA-145 overexpression and interference plasmids were in accordance with target sequences after gene sequencing.In cell proliferation and angiogenesis assays,OD value at 570 nm in miRNA-145 overexpression group was decreased（P<0.05）compared to that in control group,and miR-145 overexpression also inhibited endothelial PCNA protein and tube formation（P<0.05）.Conversely,OD value in miRNA-145 interference group was increased（P<0.01）,and miR-145 interference promoted endothelial PCNA upregulation and tube formation（P<0.01）.Meanwhile,miR-145 overexpression downregulated CREB and VEGF proteins（P<0.05）,while mi R-145 interference upregulated both of them（P<0.05）.MiR-145 directly binding to CREB3’UTR was validated by Luciferase reporter assay.Conclusion:MiRNA-145 regulates proliferation and angiogenesis of endothelial cells,which’s associated with the inhibition of miR-145 to CREB/VEGF signaling. |
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