microRNA-449a reduces growth hormone-stimulated senescent cell burden through PI3K-mTOR signaling

Abstract

Cellular senescence, a hallmark of aging, has been implicated in the pathogenesis of many major age-related disorders, including neurodegeneration, atherosclerosis, and metabolic disease. Therefore, investigating novel methods to reduce or delay the accumulation of senescent cells during aging may attenuate age-related pathologies. microRNA-449a-5p (miR-449a) is a small, noncoding RNA down-regulated with age in normal mice but maintained in long-living growth hormone (GH)-deficient Ames Dwarf (df/df) mice. We found increased fibroadipogenic precursor cells, adipose-derived stem cells, and miR-449a levels in visceral adipose tissue of long-living df/df mice. Gene target analysis and our functional study with miR-449a-5p have revealed its potential as a serotherapeutic. Here, we test the hypothesis that miR-449a reduces cellular senescence by targeting senescence-associated genes induced in response to strong mitogenic signals and other damaging stimuli. We demonstrated that GH downregulates miR-449a expression and accelerates senescence while miR-449a upregulation using mimetics reduces senescence, primarily through targeted reduction of p16^Ink4a, p21^Cip1, and the PI3K-mTOR signaling pathway. Our results demonstrate that miR-449a is important in modulating key signaling pathways that control cellular senescence and the progression of age-related pathologies.

Keywords: adipose; growth hormone; longevity; miR-449a; senescence.

Fig. 1.

Single-nuclei sequencing reveals higher percentage of fibroadipogenic precursor cells in visceral adipose tissue of df/df mice. (A) Different cell populations within visceral adipose tissue distinguished by color. Embedding is based on the 1,000 most variable genes and the first 15 harmonized principle components. Clustering was performed on the UMAP embedding. (B) Fraction (relative to the total number of nuclei) of each cell type in within visceral adipose tissue of control and df/df mice. (C) Quantification of cell populations shown in A as percentages of total population.

Fig. 2.

Fluorescence-activated cell sorting reveals higher number of adipose-derived stem cells and qRT-PCR demonstrates elevated miR-449a in total adipose tissue, adipose-derived stem cells, and preadipocytes isolated from df/df mice versus controls. (A) Total cells sorted per gram of visceral fat gated for live, lineage (−), PDGFRα (+), Sca-1 (+), and CD34 (+) populations. (B) Relative expression of miR-449a in visceral adipose tissue determined by quantitative RT-PCR. (C) Relative expression of miR-449a in sorted cells in visceral adipose tissue of N/df and df/df mice. Relative expression was calculated using the 2^−ΔΔCT method. Statistical analyses used independent t test (two groups) or one-way analysis of variance with multiple comparisons (Tukey’s test). Values depicted as mean ±SEM. *P value < 0.05, **P value < 0.01, ***P value < 0.001.

Fig. 3.

Senescence-associated β-galactosidase activity is suppressed in miR-449a mimic-HUVECs. (A) Percentage of senescence-positive cells in control, miR-449a mimic-transfected, and miR-449a inhibitor-transfected HUVECs (n = 4 per group). (B) Percentage of senescence-positive (blue stained) cells in control (C), mimic-transfected (M), and inhibitor-transfected (I) HUVECs treated with 5 nM growth hormone (GH) for 10 d versus untreated controls (n = 4 per group). Blue color is produced in the presence of X-gal and β-galactosidase, wherein the enzyme β-galactosidase (highly expressed in senescent cells) cleaves X-gal to produce the observed blue color. Statistical analyses used one-way analysis of variance with multiple comparisons (Tukey’s test). Values are mean ± SEM. *P value < 0.05, **P value < 0.01, ***P value < 0.001.

Fig. 4.

miR-449a regulates pro-senescence genes and modulates the Pi3K-mTOR signaling pathway with growth hormone (GH) treatment. (A and B) Relative expression of senescence markers p21 and p16 quantified with RT-PCR in GH-treated and transfected HUVECs. (C) Relative expression of miR-449a target Ccnd1 in control, mimic-transfected, and inhibitor-transfected cells. (D and E) Relative expression of Pi3ka and mTOR in control, mimic-transfected, and inhibitor-transfected cells. (F) Relative expression of Foxo1 with GH treatment. Relative expression (n = 4 per group) was quantified through RT-PCR and calculated using the 2^−ΔΔCT method. Statistical analyses used independent t test (two groups) or one-way analysis of covariance with multiple comparisons (Tukey’s test). Values are mean ± SEM. *P value < 0.05, **P value < 0.01, ***P value < 0.001, ****P value < 0.0001.

Fig. 5.

miR-449a uptake in cocultured HUVECs secreted by adipose-derived stem cells (ADSCs) regulates pro-senescence genes and modulates Pi3K-mTOR signaling with growth hormone (GH) treatment. (A) Relative expression of miR-449a in control HUVECs, HUVECs cocultured with control ADSCs, and HUVECs cocultured with transfected ADSCs. (B–I), Relative expression of p21, Cyclin D1 (Ccnd1), Pi3ka, mTOR, Foxo1, Tnf-α, and Interleukin-1a (Il-1a) in GH-treated HUVECs cocultured with nontransfected ADSCs (control) and transfected ADSCs. Relative expression (n = 4 per group) was quantified through RT-PCR and calculated using the 2^−ΔΔCT method. Statistical analyses used independent t test (two groups) or one-way analysis of variance with multiple comparisons (Tukey’s test). Values are mean ± SEM. *P value < 0.05, **P value < 0.01.