High Glucose Promotes Osteogenic Differentiation Of Vascular Smooth Muscle Cells Through Upregulating Mircro-32 Expression Via The MAPK14-C/EBPβ Pathway

ObjectivePrevious studies of our group had found that high glucose up-regulated the expression of microRNA-32-5p(miR-32)in vascular smooth muscle cells(VSMCs)and promoted vascular calcification,but the Specific mechanism remains unclear.Thus,the aim of the current study was to investigate the regulatory mechanism of high glucose on miR-32 expression.Methods1.VSMCs were cultured in normal or high glucose condition for 7 days,and the effects of high glucose on miR-32 expression and VSMCs osteogenic differentiation were evaluated by Alkaline phosphatase(ALP)staining,qRT-PCR and Western blot(WB).2.Bioinformatics analysis and dual luciferase assay were used to analyze transcription factors that could regulate the expression of miR-32.3.Bioinformatics analysis and co-immunoprecipitation(CO-IP)were used to analyze the glucose metabolism protein kinases that could regulate miR-32 expression in VSMCs under high glucose condition.4.The effects of high glucose on the expression and phosphorylation of MAPK14 and C/EBPβ were assessed by qRT-PCR and WB after VSMCs were cultured in normal or high glucose condition for 7 days.5.SB203580 was used as a inhibitor of p-MAPK14,and the effects of SB203580 on the expression of C/EBPβ,miR-32 and on the VSMCs osteogenic differentiation were assessed by qRT-PCR and WB.6.The effects of C/EBPβ up-regulated and down-regulated on miR-32 expression and VSMCs osteogenic differentiation were assessed by qRTPCR and WB.Results1.VSMCs were cultured in high glucose condition for 7 days,and the results showed that the ALP activity was significantly increased,the mRNA and protein levels of Runx2 were significantly increased,the mRNA and protein levels of α-SMA were significantly decreased,and the expression of miR-32 expression was increased compared with the normal dose of glucose condition,.2.Bioinformatics analysis predicted that C/EBPβ was a transcription factor,which could regulate the expression of miR-32,and dual luciferase showed that miR-32 promoter activity was enhanced induced by upregulated of C/EBPβ.3.Bioinformatics analysis predicted that MAPK14 was a glycometabolic protein kinase,which could regulate miR-32 expression in VSMCs under high glucose condition,and co-immunoprecipitation(COIP)results showed that MAPK14 could interacted with C/EBPβ.4.High glucose have no significantly differences on the protein level of MAPK14.while the protein levels of p-MAPK14,C/EBPβ and pC/EBPβ were significantly increased induced by high glucose.5.SB203580 treatment have no influences on the mRNA and protein levels of MAPK14,but it can decrease the protein level of p-MAPK14,and the mRNA level,protein level and phosphorylated protein level of C/EBPβwere decreased,as well as the expression of miR-32.Furthermore,SB203580 intervention could reduce the mRNA and protein levels of Runx2,while the levels of α-SMA mRNA and protein also increased.6.When C/EBPβ overexpressed in VSMCs,the protein and phosphorylated protein levels of C/EBPβ were increased,and the level of miR-32 expression was enhanced.In addition,C/EBPβ overexpression could increase the levels of Runx2 mRNA and protein levels,and the mRNA and protein levels of α-SMA were decreased.While C/EBPβ low expression could produce the opposite effect,with decreased the expression of miR-32 and Runx2,and increased the expression of α-SMA.ConclusionHigh glucose promoted osteogenic differentiation of VSMCs through upregulating miR-32 expression,which regulated by C/EBPβ pathway.