Nrf3 Promotes Vascular Calcification Via Regulating Mir-101c

Background and Aim:During the process of vascular media calcification,the transition of vascular smooth muscle cell(VSMC)to an osteoblast-like phenotype is the fundamental pathological feature.Our group has found that Nrf3 is involved in the differentiation of embryonic stem cells into VSMC,indicating that Nrf3 may play a critical role in cardiovascular diseases.However,the role of Nrf3 in vascular calcification remains to be elucidated.In this study,we aimed to investigate the functional role of Nrf3 in vascular calcification,and unravel its underlying molecular mechanisms.MethodsPrimary mouse vascular smooth muscle cells were isolated and cultured in vitro.We cultured VSMC with high concentrations of inorganic calcium and phosphorus to induce smooth muscle cell calcification.This in vitro model simulates patients with chronic kidney disease(CKD)with hypercalcemia and hyperphosphatemia.We then overexpressed or inhibited Nrf3 activity to find out its function in the phenotypic switching of vascular smooth muscle cells into osteogenic cells.MiR-101 c is identified as the functional target of Nrf3 by chromatin immunoprecipitation sequencing(CHIP-seq)and Dual-Luciferase Reporter Assay.The role of MiR-101 c in VSMC calcification were then assessed using overexpression and knockdown techniques.In vivo,high-dose of vitamin D were administered subcutaneously for three days to induce vascular calcification in order to explore the role of Nrf3 in the VD-induced vascular calcification.Finally,we collected the calcified blood vessels and non-calcified blood vessels of CKD patients to explore the different expression of Nrf3.ResultsThe expression of Nrf3 was significantly increased during the SMCs calcification.Knocking down Nrf3 was able to reduce cell calcification.Conversely,SMCs calcification was significantly increased after overexpressing Nrf3.MiR-101 c was identified as the functional target gene based on the CHIP-seq and Dual-Luciferase Reporter Assay.Overexpression of Nrf3 inhibits the expression of miR-101 c at theRNA level,while inhibition of Nrf3 increases the expression of miR-101 c.Dual-Luciferase Reporter Assay confirmed this result.Next,we studied the effect of miR-101 c on the calcification of VSMC.In the process of VSMC calcification in vitro,the expression of miR-101 c gradually decreased.Inhibiting the expression of miR-101 c significantly aggravated the calcification,and vice versa.By searching the database,we first predicted map3k1,akt2,and msx2 as potential targets of miR-101 c.Through a series of molecular experiments and dual luciferase report experiments,we found that map3k1 is one of the targets of miR-101 c but not akt2 or msx2,via phosphorylation of map3k1/ERK.The mechanism of regulating SMCs calcification is inhibition of phosphorylation of map3k1/ERK.In vivo experiments,usingRNA sequence,similar differences on the erk signaling pathyway was also found in our in vivo experiments.Then,vitamin D was used to induce vascular calcification.The expression of miR-101 c in the aorta was increased in the Nrf3 knockout(KO)mice and protected from vascular calcification compared with wildtype(WT)mice.Immunofluorescence staining of the aorta showed that the fluorescence intensity of p ERK and RUNX2 in Nrf3 KO mice was markedly lower than the control group.ConclusionsThis study revealed the role of Nrf3 in vascular calcification.Nrf3 could promote VSMC osteogenic phenotype transformation to aggravate vascular calcification.MiR-101 c is a functional target of Nrf3.Nrf3 can directly bind to the downstream region of miR-101 c and inhibit the expression of miR-101 c.MiR-101 c can attenuate vascular calcification by inhibiting the phosphorylation of Map3k1/ERK and down-regulating the expression of RUNX2.It is suggested that Nrf3/miR-101 c may be a new target for the treatment of vascular calcification-related diseases.