Aging is a major driving factor for the decline of vascular functions. Over time, senescent endothelial cells accumulate, which promote endothelial dysfunction and cardiovascular disease (CVD). MicroRNAs play a central role in CVD and cellular senescence.
MicroRNA-375-3p expression levels were analyzed in non-senescent and senescent human coronary artery endothelial cells (HCAEC) by qRT-PCR. To assess its cellular effects, miR-375-3p was downregulated by transfection with a LNA-inhibitor. In silico predicted targets of miR-375-3p were validated on mRNA level.
A machine learning analysis of 1800 miRNAs from over 1200 datasets of various tissue samples from healthy individuals aged 20 to 90 years showed that miR-375-3p is upregulated in aged individuals, exhibiting a high centrality and targeting over 80% of the respective age-associated targets. Expression analyses showed a strong upregulation of miR-375-3p in senescent versus non-senescent HCAEC (p<0.05) and in aortic endothelial cells of old compared to young mice. Successful inhibition of miR-375-3p improved angiogenic capacities as shown in tube formation and sprouting assays (p<0.05). MiR downregulation decreased the expression of senescence-associated beta-galactosidase (p<0.05). However, cell death was elevated and proliferation hampered in non-senescent HCAEC (p<0.05). Furthermore, levels of inflammatory cytokines were reduced in non-senescent and increased in senescent HCAEC (e.g. IL-6, p<0.05). In silico, THBS1 and NUCB2 were identified as potential targets of miR-375-3p and their regulation was confirmed by qRT-PCR (p<0.05, respectively).
A machine-learning based analysis identified microRNA-375-3p as a key microRNA in aging. Its upregulation was confirmed in vitro and in vivo. Inhibition of miR-375-3p improved angiogenic capacities in non-senescent and replicative senescent HCAEC, proposing a positive effect on vascular regeneration. Future experiments will focus on the effect of the identified target genes and miR-375-3p in cellular aging.
