Rno-miR-29b-3p/MMP2 Regulate Rat Arterial Calcification Study

Part one:The Involvement of mi R-29b-3p in Arterial Calcification by Targeting Matrix Metalloproteinase-2Aim: To investigatethe effect of miRNA-29b-3p on MMP2 expression and arterial calcification.Materials and Methods: 20 male SD rats were divided randomly into two groups,control and calcification group,10 rats every group.In calcification group,high dose of vitamin D3(60 million U/kg/day)was used to inject at back subcutaneous of rats and in control group,rats were injected with equal dose of saline,3 consecutive days.All rats were killed after 10 days and thoracic and abdominal aorta were immediately separated and parts arterial tissues were stored with 4% paraformaldehyde for making tissue sections and the others were stored liquid nitrogen.HE staining was used to observed two group rats arterial tissues sections structure,and Von kossa staining was used observed two group rats arterial tissues sections calcium deposition.mimics and inhibitors of miRNA-29b-3p were transfected rat A7R5 cells to overexpress and silent miRNA-29b-3p.q RT-PCR and Western blotmeasured miRNA-29b-3p and MMP2 m RNA levels and protein levels both in arterial tissue and cells.Pearson linear least squares regression analyses analyze correlation between miRNA-29b-3p and MMP2 m RNA and protein levels in arterial tissue.Bioinformatics predicted target genes of miRNA-29b-3p.Double luciferase reporter assay measured the directly influences miRNA-29b-3p in MMP2 expression.Results:HE staining showed aortic wall structure damage,elastic fiber degradation,disorderly arrangement in calcification group and aortic wall structure is complete in control group.Von Kossa staining shows black calcium was deposited in tunicae media along with elastic fiber in calcification and there was no calcium deposition in the normal control group.q RT-PCR results demonstrated miRNA-29b-3p in calcification group was significantly lower than control group and MMP2 in calcification group was higher than control group.Pearson linear least squares regression analyses suggested there was a negative correlation between miRNA-29b-3p expression and MMP2 m RNA expression(P=0.0014,R2=0.481)and MMP2 protein expression(P=0.0026,R2=0.443).The results of transfection of miRNA-29b-3p mimics and inhibitors showed miRNA-29b-3p up-regulation and down-regulation leaded to MMP2 expression decreasing and increasing.Bioinformatics predicting target genes suggested MMP2 may be a target genes of miRNA-29b-3p.Double luciferase reporter assayshowed miRNA-29b-3p plays a directly influences on MMP2 expression.Conclusion: First,miRNA-29b-3p expression is decreasing and MMP2 expression is increasing in vitamin D3 induced rat arterial calcification model.Second,MMP2 is a target gene of miRNA-29b-3p.Third,miRNA-29b-3p is involved in arterial calcification may through regulating MMP2 expression.Part two miRNA-29b-3p /MMP2 inhibits VSMCs transdifferentiated osteoblast-like cells and calcification in vitroAim: To investigate the effect of miRNA-29b-3p / MMP2 on phenotypic transformation and calcification of vascular smooth muscle cells.Materials and Methods:Co-transfection with lentivirus packaging plasmid,PRSV\PMDLG\PMD2 and miRNA-29b-3p overexpressing plasmids or silencing plasmids or scramble plasmids in 293 T cells.Virus,293 T cells producing,were used to infected rat VSMCs to construct miRNA-29b-3p overexpressing and silencingstable cell lines.Fluorescence microscopy was used to observe plasmids co-transfected 293 T cells andvirus-infected VSMC.Calcification medium containing?-glycerophosphoric acidcultured VSMCs 10 days to induced calcification,including control group,calcification group,miRNA-29b-3p overexpressing group,miRNA-29b-3p silencing group and black plasmid group.Alizarin red S staining and O-Cresol Phthalocyanine Complex Copper Colorimetry measured each group VSMCs calcification.q RTPCR was used to detected miRNA-29b-3p and MMP2 and other osteogenic differentiation markers Runx2 and Msx2.Western blot was used to measured MMP2 protein levels.Results:miRNA-29b-3p overexpressing and silencing stable VSMC lines were succeeding in establishing with lentivirus.Overexpression and silence of miRNA-29b-3p in VSMCs resulted in decreasing and increasing of MMP2,respectively.Overexpression of miRNA-29b-3p in VSMCs decreased calcium deposition and inhibited VSMC osteogenesis differentiation maker genes Runx2 and Msx2 expression and calcification in ?-glycerophosphoric acid calcification medium compared with scramble group,while silence of miRNA-29b-3p in VSMCs promoted VSMCs osteogenic markers expressionand calcification.Conclusion:miRNA-29b-3p inhibit VSMCs Osteogenesis differentiation and calcification in?-glycerophosphoric acidcalcification environment,at least partly by targeting MMP2.