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. 2014 Sep;6(9):771-87.
doi: 10.18632/aging.100693.

Age- and glycemia-related miR-126-3p levels in plasma and endothelial cells

Fabiola Olivieri  1 , Massimiliano Bonafè  2 , Liana Spazzafumo  3 , Mirko Gobbi  4 , Francesco Prattichizzo  4 , Rina Recchioni  5 , Fiorella Marcheselli  5 , Lucia La Sala  6 , Roberta Galeazzi  7 , Maria Rita Rippo  4 , Gianluca Fulgenzi  4 , Sabrina Angelini  8 , Raffaella Lazzarini  4 , Anna Rita Bonfigli  9 , Francesca Brugè  10 , Luca Tiano  10 , Stefano Genovese  11 , Antonio Ceriello  6 , Massimo Boemi  9 , Claudio Franceschi  12 , Antonio Domenico Procopio  1 , Roberto Testa  13
Affiliations

Age- and glycemia-related miR-126-3p levels in plasma and endothelial cells

Fabiola Olivieri et al. Aging (Albany NY). 2014 Sep.

Abstract

Circulating miR-126-3p levels were determined in 136 healthy subjects (CTRs) aged 20-90 years and 193 patients with type-2 diabetes mellitus (T2DMs) aged 40-80 years, to explore the combined effect of age and glycemic state on miR-126-3p expression. Moreover, intra/extracellular miR-126-3p levels were measured in human endothelial cells (HUVECs) undergoing senescence under normo/hyper-glycemic conditions. Plasma miR-126-3p was significantly higher in the oldest compared with the youngest CTRs ( <45 vs. >75 years; relative expression: 0.27±0.29 vs. 0.48±0.39, p=0.047). Age-based comparison between CTRs and T2DM demonstrated significantly different miR-126-3p levels only in the oldest (0.48±0.39 vs. 0.22±0.23, p<0.005). After multiple adjustments, miR-126-3p levels were seen to be lower in patients with poor glycemic control, compared with age-matched CTRs. The age-related increase in plasma miR-126-3p found in CTRs was paralleled by a 5/6-fold increase in intra/extracellular miR-126-3p in in vitro-cultured HUVECs undergoing senescence. Notably, significant down- regulation of SPRED-1 protein, a validated miR-126-3p target, was found in senescent HUVECs. Moreover, miR-126-3p expression was down-regulated in intermediate-age HUVECs grown in high-glucose medium until senescence. Aging/senescence-associated miR-126-3p up-regulation is likely a senescence-associated compensatory mechanism that is blunted when endothelial cells are exposed to high glucose levels, a phenomenon that probably occurs in vivo in T2DM patients.

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Conflict of interest statement

Conflict of interest statement

The authors of this manuscript declare no conflict of interest.

Figures

Figure 1
Figure 1. Relative miR-126-3p expression in plasma from 136 healthy subjects (CTR) and 193 patients (T2DM) divided into age groups
Error bars (a) showing relative miR-126-3p expression (in a.u.) in plasma of young (20-45 years, n=44), elderly (46-74 years, n=57) and old (≥ 75 years, n=34) CTR and in plasma of T2DM subjects divided into two age groups (46-74 years, n=155 vs. ≥ 75 years, n=38). *p from t-test with Bonferroni correction for multiple comparisons. ANOVA, F= 4.887, p=0.001. Scatter plots (b and c) showing relative miR-126-3p expression (in arbitrary units, a.u.) according to age in CTR subjects (b) and T2DM patients (c). Data are expressed as mean of 2ΔΔΔCt normalized with cel-miR-39.
Figure 2
Figure 2. Relative miR-126-3p expression in 92 CTR and 193 T2DM subjects
Standardized means of relative miR-126-3p expression (in a.u.) in 193 T2DM patients and 136 CTR subjects divided into age groups (T2DM: 46-74 years and ≥ 75 years; CTR: 20-45 years, 46-74 years and ≥ 75 years).
Figure 3
Figure 3. Relative miR-126-3p expression in 92 CTR and 193 T2DM subjects divided into those with good and poor glycemic control based on HbA1c levels
Relative miR-126-3p expression (in arbitrary units, a.u.) in 92 CTR subjects and T2DM patients divided into those with good (GGC, n=106) and poor glycemic control (PGC, n=87) based on an HbA1c cut-off of 7% (53 mmol/mol) (a). Data are expressed as 2ΔΔΔCt normalized with cel-miR-39. *GLM, adjustment for age, platelet count and ApoAI levels, p<0.05.
Figure 4
Figure 4. Characterization of young, intermediate-age and senescent HUVECs
Bar chart showing cumulative population doubling (CPD) (a), SA-β-gal activity (%) (b), p16INK4a relative expression reported as fold changes vs. young cells (c) and telomere length (T/S) reported as fold changes vs. young cells (d), in young, intermediate age and senescent HUVECs. *GLM, p<0.05.
Figure 5
Figure 5. Relative miR-126-3p expression and SPRED-1 protein levels in young, intermediate age and senescent HUVECs
Young, intermediate age and senescent HUVECs: intracellular miR-126-3p (a) and miR-126-3p recovered from conditioned medium (b). Data are expressed as 2−ΔCt normalized with RNU44, and reported as arbitrary units (a.u.). Western blot and densitometric analysis of SPRED-1 (c and d, respectively) were performed in the same samples. Data from three independent experiments are expressed as percentage of intensity in young cells. *GLM, p<0.05.
Figure 6
Figure 6. Relative miR-126-3p expression and SPRED1-protein levels in intermediate age HUVECs undergoing senescence in normoglyacemic and hyperglycemic conditions
Intracellular miR-126-3p and miR-126-3p recovered from medium in normoglycemic (NG) (a) and hyperglycemic (HG) cultures (b). SPRED1-protein levels in normoglycemic (NG) and hyperglycemic (HG) cultures (c). Hyperglycemic medium contained 25 mM glucose. Mannitol was used as an osmotic control in the normoglycemic cultures. MiR-126-3p is expressed as 2ΔΔΔCt normalized with RNU44. * GLM, p<0.05.
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Comment in

  • Circulating microRNAs in aging.
    Bonafè M, Olivieri F. Bonafè M, et al. Oncotarget. 2015 Jan 30;6(3):1340-1. doi: 10.18632/oncotarget.3175. Oncotarget. 2015. PMID: 25633812 Free PMC article. No abstract available.

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