The Role And Mechanism Of MiR-32-5p In The Regulation Of PTEN Expression In Mouse Vascular Smooth Muscle Cells

Objective: This research intends to explore miR-32-5p target gene and cell signaling pathway,in order to provide experimental evidence for the effective potential intervention of vascular calcification.Methods: 1.The mature sequence of miR-32-5p was obtained through the online nucleic acid database,then used the target gene prediction software and combined with the relevant factors to screen out the target gene of miR-32-5p.2.Dual-Luciferase Reporter Assay System was carried out to investigate if the gene was the direct target of miR-32-5p.3.MVSMCs were tansfected with miR-32-5p mimic and miR-32-5p inhibitor or the respective control,and the level of miR-32-5p was determined by qRT-PCR.4. qRT-PCR and Western blot analysis were used to evaluated the expression of mRNA and protein expression level of the target gene.5.Western blot analysis were used to evaluate the change of related downstream proteins.Results: 1. miRanda, TargetScan, RNAhybrid and other databases were used to find out PTEN was the one of target genes ofmiR-32-5p,combined with bioinformatics and literature analysis.2.Overexpression of miR-32-5p by tansfection with miR-32-5p mimic decreased PTEN protein levels in MVSMCs,while miR-32-5p inhibition by tansfection of miR-32-5p inhibitor significantly elevated PTEN protein levels. But did not significantly affect the expression level of PTEN mRNA when tansfected with miR-32-5p mimic or miR-32-5p inhibitor in MVSMCs.3.DNA sequencing analysis showed that the WT-GV272-PTEN vector,MUT-GV272-PTEN vector were successfully constructed. Dual-luciferase activity cotransfected with miR-32-5p mimic and WT-3'UTR of PTEN were downregulated in MVSMCs compared to NC group,whlie Dual-luciferase activity cotransfected with miR-32-5p inhibitor and WT-GV272-PTEN were up-regulated in MVSMCS compared to NC group.4.Western blot showed that the ectopic expression of miR-32-5p could induce the phosphorylation of Akt, leading to the increased phosphorylation and expression of Runx2 and the inhibition of Akt phosphorylation could abrogate miR-32 induced phosphorylation and expression of Runx2.Conclusion:1.miR-32-5p inhibited the expression of PTEN by targeting PTEN 3'UTR sequence in MVSMCs, PTEN was confirmed as a target gene of miR-32-5p in MVSMCs.2.miR-32-5p might promote osteoblastic transdifferentiation of MVSMCs by increasing the expression and phosphorylation of Runx2 through targeting 3'UTR ofPTEN, and subsequently activating PI3K-Akt signaling pathways.