High Glucose Promotes VSMCs Calcification Via Up-regulating MiR-32 To Targetedly Inhibit GATA6

ObjectiveTo clarify the function and mechanism of high glucose in promoting calcification of vascular smooth muscle cells(VSMCs)by up-regulating microRNA-32-5p(miR32)to reveal a new signaling pathway of vascular calcification in diabetes.Methods1.β-glycerol phosphate disodium(β-GPD)with final concentration of 10 mM was added to medium with normal(5 mM)and high(25 mM)glucose concentration respectively to induce VSMCs calcification for establishing the calcification model.qRT-PCR was used to test the expression level of miR-32.The expression levels of osteogenesis marker genes(ALP,RUNX2)and smooth muscle marker genes(α-SMA,SM22α)were detected by qRT-PCR and western blot(WB).The formation of calcium nodules were observed by alizarin red staining.2.The expression of osteogenesis marker genes and smooth muscle marker genes were detected by qRT-PCR and WB after mimic NC or miR-32 mimics transfected into VSMCs.The formation of calcium nodules were observed by aizarin red staining.3.The possible target gene GATA6 of miR-32 in the process of vascular calcification was screened by using databases such as miRanda,Target Scan,RNAhybird.The correlation between miR-32 and GATA6 was verified by dual luciferase reporter gene assay.The expression of GATA6 in calcification model was detected by qRT-PCR and WB.4.GATA6 overexpressed lentivirus was transfected into VSMCs.qRT-PCR and WB were used to detect the efficiency of GATA6 lentivirus efficiency and the expression of osteogenesis marker genes and smooth muscle marker gene.The formation of calcium nodules were observed by alizarin red staining.5.Lentiviral of GATA6 siRNA was transfected into VSMCs.WB was used to detect the expression of osteogenesis marker genes and smooth muscle marker gene.6.GATA6 overexpression lentivirus and miR-32 mimic were co-transfected into VSMCs.The expression of osteogenesis marker gene,smooth muscle marker gene and GATA6 were detected by WB.The formation of calcium nodules were observed by alizarin red staining.7.The potential downstream target genes sm-MHC of GATA6 in the process of vascular calcification was obtained by screening databases.Dual luciferase reporter gene assay and chromatin immunoprecipitation(ChIP)were used to verify their correlation.WB was used to verify the expression level of sm-MHC in calcification model.Results1.The results of calcification model showed that :(1)After high glucose treatment,the expression of osteogenic differentiation marker genes ALP and RUNX2 were significantly up-regulated,while the expression of vascular smooth muscle marker genes α-SMA and SM22αwere significantly down-regulated.Alizarin red staining showed that high glucose promoted the formation of calcified nodules.(2)The expression of miR-32 gene expression significantly increased in the high glucose treatment group,especially in the high glucose plus calcification inducer group.2.The expression of osteogenic differentiation marker genes ALP and RUNX2 were obviously incresead while the expression of vascular smooth muscle marker genes α-SMA and SM22α were obviously decreased after transfecting miR-32 mimic into VSMCs.Alizarin red staining showed that miR-32 transfection promoted the formation of calcified nodules in VSMCs.3.Dual luciferase reporter gene assay indicated that GATA6 was the target gene of miR-32 and the expression of GATA6 was down-regulated in the high glucose plus calcification inducer group.4.The expression of osteogenic differentiation marker genes,ALP and RUNX2,were significantly decreased,while the expression of smooth muscle marker gene α-SMA significantly increased after GATA6 overexpression lentivirus were transfected into VSMCs.Aizarin red staining showed that the overexpression of GATA6 inhibited the formation of calcium nodules.5.After GATA6 siRNA transfected into VSMCs,the expression of osteogenic differentiation marker genes ALP and RUNX2 were significantly up-regulated while the expression of vascular smooth muscle marker gene α-SMA was significantly down-regulated.6.Co-transfection of GATA6 overexpression lentivirus and miR-32 mimic to VSMCs confirmed that GATA6 overexpression could antagonize miR-32-induced VSMCs calcification.7.Dual luciferase reporter gene assay and ChIP test results showed that GATA6 can bind to the sm-MHC promoter region.ConclusionHigh glucose promotes VSMCs calcification via up-regulating miR-32 to inhibit GATA6.