The MiR-182/Sort1 Axis Regulates Vascular Smooth Muscle Cell Calcification In Vitro And In Vivo

Part one:Sort1 regulates vascular smooth muscle cell calcification in vitro and in vivoObjective:To investigate the expression of Sort1 in calcified arteries and participate in the regulation of calcification of vascular smooth muscle cells.Materials and Methods:Male Sprague-Dawley rats were randomly divided into control and calcified groups,10 in each group.The rat aortic calcification was induced by subcutaneous injection of high-dose vitamin D3 at a dose of600,000 U/kg/day for 3 consecutive days.The same amount of normal saline was injected subcutaneously into the back of the rat as a control group.After 10days of intervention,the rats were sacrificed,the rat thoracic and abdominal aortic tissues were isolated,the surrounding connective tissue was dissected,and some of the aortic tissues were soaked in 4%paraformaldehyde for the preparation of pathological sections,and the remaining sections were stored in liquid Nitrogen for nucleic acid and protein detection.Hematoxylin-eosin（HE）and Von Kossa staining were used to compare the histopathological changes of the aorta and calcium deposition in the two groups of rats.q RT-PCR and Western Blot were used to detect the expression of Sort1 and RUNX2 mRNA and protein expression levels in the middle artery.VSMCs were calcified by 10mmol/L calcification ofβ-GP and divided into normal and calcified groups and cultured for 7 days,14 days and 21 days,respectively.The formation of calcium nodules was observed and the expression levels of Sort1 and Runx2 mRNA and protein were detected.Plasmids pRsv-REV,pMD2G,pMDLg-pRRE and Sort1silencing vector or control vector plasmids were co-transfected into 293T cells to construct a lentivirus system and infect VSMCs of rats to construct a Sort1silencing cell stabilizing strain.Inverted fluorescence microscopy was used to evaluate the efficiency of plasmid co-transfection of 293T cells and Lentivirus-infected VSMCs.The in vitro calcification model of VSMCs was induced by calcification of 10 mmol/Lβ-GP and was set as calcification group,silence group of Sort1,and blank control group,and cultured for 14 days.Alizarin red S staining was used to detect the calcification of VSMCs in each group.The expression levels of Sort1 and Runx2 mRNA and protein were detected.Results:The rat model of arterial calcification was successfully established.Compared with the aortic tissue of rats in the control group,the normal arteries of rats in the calcified group were damaged.A large number of collagen fibers and elastic fibers were disordered,and there were obvious fractures and degradations.A large amount of black calcium deposits can be seen between;β-GP successfully induced the calcification of VSMCs.Compared with the control group,the VSMCs calcification group showed obvious orange-red calcium nodules.With the prolongation of culture time,the number of orange-red calcium nodules was more and the color was more obvious;Osteoblast-like differentiation of rat VSMCs.In both in vivo and in vitro models,the expression level of RUNX2 was significantly increased at both gene and protein levels.With the prolonged calcification of VSMCs induced byβ-GP in vitro,calcium salt nodules were observed.The number of RUNX2 expressions also increased gradually;High expression of Sort1 gene and protein in rat in vivo and in vitro calcification.In in vivo and in vitro models,the expression level of Sort1 mRNA and protein Sortilin was up-regulated in calcification group,and VSMCs was calcified inβ-GP for 7 days,14 days,and 21 days.Calcium nodules increased significantly,and the expression of Sort1 mRNA and Sortilin also increased gradually;Sort1 gene plays a key role in inducing calcification and osteogenic differentiation of VSMCs.After the lentivirus silences Sort1 gene,β-GP induces osteogenic transformation and calcification of VSMCs,calcium salt nodules decrease significantly,RUNX2 expression is down-regulated,and Sort1 inhibits osteogenic differentiation and calcification through RUNX2 signaling.Conclusion:In high-dose vitamin D₃ andβ-GP can establish rat in vivo calcification model,Sort1 expression in the arterial calcification is upregulated,inhibition of Sort1 expression can inhibit osteogenic differentiation of VSMCs,inhibit the formation of calcification of VSMCs.Part two:miR-182 targets regulation of Sort1 expression in the regulation of arterial calcificationObjective:To investigate the effect of miR-182 on the expression of Sort1Materials and Methods:The bioinformatics website mi RNAbase and TargetScan were used to predict that Sort1 is a mi R-182 target gene and the structure of miR-182 was searched.Rat arterial tissue in the first part of the experiment was used to detect the expression of miR-182 in calcification and control groups by qRT-PCR.The Pearson correlation coefficient method was used to analyze the correlation between miRNA-182 and the expression level of Sort1 mRNA and protein in arterial tissue.In vitro,calcification of VSMCs was induced by calcification ofβ-GP,and the expression of miRNA-182 was detected at 7 days,14 days,and 21 days.A7r5 rat vascular smooth muscle cells were transiently transfected with miRNA-182 mimic and inhibitor and their corresponding controls.Overexpression and silencing of miRNA-182,qRT-PCR and Western Blot were used to detect the changes of miRNA-182,Sort1 mRNA and Sortilin expression levels in cells.The regulation of miRNA-182 on Sort1 targets was verified by vector construction and dual luciferase assays.Results:Through miRNAbase,TargetScan online database query,analysis and prediction of Sort1 is the target gene of miR-182,mi RNA-182 seed sequence is highly conserved in many species;miRNA-182 was highly expressed in calcified arteries in rats.Pearson’s correlation coefficient method was used to analyze the negative correlation between miRNA-182 and the expression levels of Sort1 mRNA and Sortilin in arteries;In theβ-GP-induced in vitro cell calcification model,the expression of miRNA-182 after 14 days was significantly lower than that of the control group;The transient transfection results of miRNA-182 mimic and inhibitor showed that the increase or decrease of miRNA-182 expression could reduce or increase the expression level of Sort1protein;Dual luciferase reporter assays verify that Sort1 is the target gene for miRNA-182.Conclusion:mi RNA-182 is down-regulated in arterial calcification in high-dose vitamin D₃ andβ-GP can establish rat in vitro calcification model.Over-expression or silencing of miRNA-182 down-regulates or up-regulates Sort1,and miRNA-182 has direct regulation effect on Sort1.