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P11 Role of MIR-145 as a mediator of DNA damage and senescence in vascular smooth muscle cells from type 2 diabetes patients
  1. KE Hemmings1,
  2. K Riches1,
  3. DJ O’Regan2,
  4. NA Turner1,
  5. KE Porter1
  1. 1Division of Cardiovascular and Diabetes Research, Leeds Institute of Cardiovascular and Metabolic Medicine (LICAMM), University of Leeds, UK
  2. 2Department of Cardiac Surgery, Yorkshire Heart Centre, Leeds General Infirmary, Leeds, UK

Abstract

Introduction Type 2 diabetes (T2DM) confers a significant risk of premature coronary heart disease and poor outcomes after saphenous vein (SV) bypass grafting. We previously reported an aberrant phenotype in smooth muscle cells (SMC) cultured from T2DM patients, and elevated expression levels of a small non-coding RNA, microRNA (miR)-145. Here we explored the ?relationship between miR-145 overexpression and the senescence- associated secretory phenotype (SASP).

Methods Cultured SMC from non-diabetic (ND) and T2DM patients were investigated for markers of senescence (SA β-gal), DNA damage (γH2AX immunofluorescence) and expression of SASP molecules (real time RT-PCR). In some experiments, ND-SMC were exposed to a DNA damaging stimulus, (etoposide 5?µM) and in others, miR-145 expression was manipulated via transfection of pre-miR- or anti-miR-145, as appropriate.

Results Native T2DM-SMC displayed increased levels of senescence (n = 7, p < 0.01) and γ-H2AX staining (n = 8, p < 0.01). Knockdown of miR-145 partially reversed these features (n = 6, p < 0.05 and p = 0.07, respectively). Exposure of ND cells to etoposide or oxidative stress led to increased miR-145 expression (n = 6, P < 0.05). Conversely overexpression of miR-145 in ND cells induced a significant increase in senescence (P < 0.01), a trend towards increased DNA damage (P = 0.08), elevated expression of MCP-1 (P < 0.05) and a tendency towards increased TGFβ3 expression (n = 3 for both).

Conclusion T2DM SV-SMC are aberrant in terms of senescence, DNA damage and expression of miR-145. Overexpression of miR-145 in ND-SMC increases senescence, DNA damage and secretion of SASP molecules, particularly MCP-1. Delineation of the role of miR-145 as a cause or effect of DNA damage is an important aim.

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