Downregulation of miR-342-3p/miR-342-5p in type 1 diabetes mellitus and vascular health; Case controlled study in peripheral blood mononuclear cells and plasma, CD45dimCD34+CD133+ cells, inflammatory markers, CXCR1/2 and PANEX2 mRNA combined with Ingenuity Pathway Analysis

Abstract BACKGROUND Cardiovascular disease (CVD), the leading causes of death worldwide, correlates with inflammation and shortens life span in type 1 diabetes (T1DM). In animals, miR-342 were shown to be anti-inflammatory in CVD. We hypothesise: proangiogenic miR-342-3p/-5p are downregulated in T1DM whereas inflammatory cytokines are upregulated.METHODS We studied plasma/peripheral blood mononuclear cells (PBMCs) in 29 T1DM and 20 controls (HC). Vascular health was measured by Fibronectin Adhesion Assay (FAA), CD45 dim CD34 + 133 + cells (flow cytometry) and Tissue inhibitor of metalloproteases (TIMP-1). Inflammatory markers IL-7, IL-8, TNFα and VEGF-C were assayed by Mesoscale assay, mRNA for chemokine receptor CXCR1/2, PANX2 and miR 342-3p/5p by qRT-PCR. Analysis: Unpaired Student t test, Pearson correlation and Ingenuity Pathway Analysis (IPA) to predict miR-342-3p/-5p targets.RESULTS In T1DM, pro-inflammatory cytokines IL-8 and TNF-α, IL-7 and growth factor VEGF-C were increased versus HCs; p=0.008, p=0.003, p=0.041 and p=0.013 respectively. MiR-342-3p/-5p were downregulated in PBMCs in T1DM versus HC; p=0.01, but not in plasma. PANX2, CXCR1 and CXCR2 mRNA were increased in PBMCs in T1DM versus HCs; p=0.004, p=0.017 and p<0.001 respectively. MiR-342-3p correlated negatively with TIMP-1, IL-6, IL-8, TNF-α, HbA 1c ; p=0.006, p=0.031, p=0.029, p=0.038, p=0.001, p=0.031 whilst miR-342-5p with TIMP-1, IL-6, IL-8, HbA 1c , p=0.005, p=0.034, p=0.029, p=0.001, p=0.042 respectively. There was positive correlation between miR-342-3p and FAA, CXCR1, CXCR2; p=0.006, p=0.019, p=0.001 and between miR-342 - 5p and FAA, CXCR2; p=0.038, p=0.036 respectively. IPA predicted miR-342-3p/-5p to be anti-inflammatory through the indirect regulation of mitogen-activated protein 3 kinase 1, inhibitor of nuclear factor kappa B kinase regulatory subunit Gamma, Surfactant protein A1 and PANX2 as well as predicting IL-8, TNF-α, IL-7 and glucose to activate the inflammatory response. ROC curve analyses showed: miR-342-3p/-5p to be a biomarker for T1DM: p=0.01, p =0.006 respectively (2) significant downregulation of miR-342-3p/-5p has occured at HbA 1c > 46.45 mmol/mol, p=0.006, p =0.005 respectively.CONCLUSION Our findings validated animal studies. Downregulation of miR-342-3p/-5p by hyperglycaemia and increase in cytokines confirmed inflammation in T1DM. MiR-342-3p/5p correlated with indices of vascular health and defined early CVD at HbA1c of 46.5 mmol/mol cut off point. MiR-342-3p/-5p based either intervention or monitoring may prove to be beneficial in CVD in T1DM.