The Hyper-methylation Of MiR-145 Promoter Is The Key Regulator For CD137 Activation And Plaque Formation

Background:Atherosclerosis?AS?is a chronic inflammatory disease caused by a variety of factors such as vascular injury,innate immunity and lipid infiltration.The terminal event is plaque formation and rupture,which can cause myocardial infarction and instability Angina,stroke,peripheral thrombosis and other cardiovascular and cerebrovascular adverse events,seriously endangering human health.MiR-145 is a microRNA that has a protective role in cardiovascular disease.It has been demonstrated that there is a persistent decrease of miR-145in the vascular wall of AS,but the potential mechanism remains unclear.This study aimed to elucidate the regulatory mechanism of mi R-145 promoter methylation status in tissues and cells at the DNA level.In this article,we further investigate the role of miR-145 promoter aberrant methylation in the development of AS plaques.Objectives:1.To detect the methylation status of mi R-145 promoter in plaques and TNF-?-treated smooth muscle cells.2.Screening the corresponding methylase or protein involved in the regulation of miR-145methylation.3.Observing miR-145 expression changes in vitro and in vivo treated with 5-aza.4.Finding the downstream target of miR-145 involved in AS progression.Materials and MethodsEthics statement and tissue samplesMale,eight week-old ApoE^-/-mice were divided into 0,6,12,18,24 weeks group,fed with western diet for recording months and euthanized through CO₂ anaesthesia.Aortas were removed and the part from root to ascending aortas was cross-sectioned for staining,the other parts were used for DNA or RNA or protein extraction.Cell culture and treatmentMouse VSMCs were isolated from aorta of male,eight-week-old C57BL/6J mice.Cells were cultured in DF12 containing 10%FBS at 37?with 5%CO₂.According to related experiments,cells were treated with TNF-??10ng/ml?for 24 hours or TNF-?plus 5-aza?5?mol/ml?for 48 hours.In CD137 associated assay,VSMCs were pretreated with TNF-??10ng/ml?for 24 hours to elevate the CD137 on membrane and further treated with recombinant CD137L?10?g/ml?to activate CD137 signaling.5-aza treatment of ApoE^-/-miceEight week-old ApoE^-/-mice were fed with western diet for 12 weeks to initiate the plaque formation and then divided into 5-aza group and saline group.C57BL/6 mice were fed with western diet for 24 weeks as WT group.Mice were received saline or 5-aza injection ip?0.25 mg/kg body weight,3 times per week?for another 12 weeks.Bisulfite genomic sequencing?BSP?and Methylation-Specific PCR?MSP?DNA was extracted from cells or tissues and performed bisulfite conversion.After conversion,DNA was used in MSP for CpG site or sent to Sangon?Shanghai,China?for BSP detection.Histology,immunohistochemistry and immunofluorescence.Masson staining was used to observe the fibrous cap in plaques.The plaque size and necrotic core were measured.Immunohistochemistry of Mac3 was performed to indicate inflammation degree in plaques.Western blotWestern blot were used to detect CD137,DNMT3a and DNMT3b;TET2,DNMT1 and NFATc1 proteins in vivo and in vitro.Bands were detected by enzyme-linked chemiluminescence.Chromatin immunoprecipitation?ChIP?assay on miR-145 promoterChromatin binding to DNMT1 and TET2 proteins was extracted using ChIP assay kit.DNA was acquired and detect level of miR-145 promoter level through Q-PCR and PCR agarose gel electrophoresis.MiRNA extraction and real-time quantitative PCRTotal RNA or miRNAs were extracted from cells or tissue samples with TRIzol.cDNA synthesis for mRNA or mi R-145 was performed using Thermo Fisher RT reagents or miRNA First Strand cDNA Synthesis kit?Sangon,China?respectively.Vectors,cell culture,and treatmentThe NFATc1 and CD137 overexpression vector with/without the 3'UTR were constructed into the p3×FLAG-CMV-24 vector.The TET2 or NFATc1 overexpression and sh-NFATc1,sh-DNMT1 knock down sequences were inserted into the Plenti-puro or PLKO.1vector respectively to created overexpression or knock-down plasmid.Luciferase reporter assayPsicheck2 reporter vectors containing the 3-UTRs of NFATc1 or CD137 and their mutants were co-transfected with mimic/inhibitors into 293T cells.At 48h after transfection,luciferase activity was determined.Results:1.The expression of mi R-145 in ApoE^-/-mice decreased with the plaque progressionMasson staining revealed a gradual increase of plaque contents with necrotic core and the Mac3 staining was used as a marker for plaque inflammation.Q-PCR showed that the level of miR-145 in the tissues decreased with the progression of plaques.2.The hyper-methylation of miR-145 promoter in plaques and VSMC stimulated by TNF-?BSP and MSP showed that the methylation level of miR-145 promoter increased with the progression of plaque.In addition,TNF-?which mimics the inflammatory environment,can also induce the hyper-methylation of miR-145 promoter in VSMC.3.DNMT1 and TET2 mediate aberrant methylation of the miR-145 promoter in inflammationThrough screening methylation-related proteins,DNMT1 and TET2 showed significant changes both in the expression and activity in plaques and VSMCs stimulated with inflammation cytokine,whereas the hyper-methylation of miR-145 promoter could be reversed through DNMT1 silencing or TET2 overexpression,which in turn lead to mi R-145elevated.4.CD137/NFATc1 pathway is downstream target gene of miR-145By bioinformatics prediction,we found that CD137/NFATc1 may be the downstream target gene of miR-145.MiR-145 mimics or inhibitors transfected into VSMCs can significantly reduce or increase the expression of both CD137/NFATc1,respectively.It was further clarified by constructing vectors for the corresponding target sites.Conclusion:DNMT1 elevation and TET2 decline caused abnormal hyper-methylation of mir-145 in plaques,which activated CD137/NFATc1 signaling and induced plaque formation.