MicroRNA- 19a- 3p Decreases with Age in Mice and Humans and Inhibits Osteoblast Senescence

Abstract

Aging is a major risk factor for most chronic diseases, including osteoporosis, and is characterized by an accumulation of senescent cells in various tissues. MicroRNAs (miRNAs) are critical regulators of bone aging and cellular senescence. Here, we report that miR-19a-3p decreases with age in bone samples from mice as well as in posterior iliac crest bone biopsies of younger versus older healthy women. miR-19a-3p also decreased in mouse bone marrow stromal cells following induction of senescence using etoposide, H₂O₂, or serial passaging. To explore the transcriptomic effects of miR-19a-3p, we performed RNA sequencing of mouse calvarial osteoblasts transfected with control or miR-19a-3p mimics and found that miR-19a-3p overexpression significantly altered the expression of various senescence, senescence-associated secretory phenotype-related, and proliferation genes. Specifically, miR-19a-3p overexpression in nonsenescent osteoblasts significantly suppressed p16 ^Ink4a and p21 ^Cip1 gene expression and increased their proliferative capacity. Finally, we established a novel senotherapeutic role for this miRNA by treating miR-19a-3p expressing cells with H₂O₂ to induce senescence. Interestingly, these cells exhibited lower p16 ^Ink4a and p21 ^Cip1 expression, increased proliferation-related gene expression, and reduced SA-β-Gal+ cells. Our results thus establish that miR-19a-3p is a senescence-associated miRNA that decreases with age in mouse and human bones and is a potential senotherapeutic target for age-related bone loss. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Keywords: AGING; CELLS OF BONE; OSTEOBLASTS.

Fig. 1

Differentially expressed age‐related miRNAs. (A) Schematic diagram of design for miRNA sequence data in mice (designed using Biorender.com). (B) miRNA sequence data showing miRNAs with log fold‐change > [1] that were differentially expressed in bone samples from vertebrae of young (6‐month) and old (24‐month) male mice (n = 10/group). The bars colored in blue highlight the miRNAs that were predicted to regulate the cellular senescence pathway using in silico analyses. (C) Normalized enrichment score (NES) for miRNAs differentially expressed with aging and predicted to target cellular senescence pathway (blue bars in B). (D) Schematic diagram of design for human bone biopsies from posterior iliac crest (designed using Biorender.com). (E) RT‐qPCR expression of miRNAs predicted to regulate cellular senescence was assessed in needle bone biopsies from posterior iliac crest of young (mean ± SD; 27 ± 3 years) and old (mean ± SD; 78 ± 6 years) healthy female volunteers (n = 10/group). Values of p are shown numerically with p < 0.05 (independent samples t test).

Fig. 2

Change in expression of miR‐19a‐3p with senescence. (A) Schematic diagram of design showing induction of senescence following DNA damage (designed using Biorender.com). Representative images of SA‐β‐Gal‐stained BMSCs treated with (B) vehicle (DMSO) and etoposide (20 uM), (C) control (untreated) and H₂O₂, (D) early and late passage (magnification ×10; n = 3/group). RT‐qPCR analysis of (E–G) p16 ^Ink4a and p21 ^Cip1 and (H–J) miR‐19a‐3p in nonsenescent and senescent BMSCs. Gene expression was denoted as fold‐change relative to vehicle or control (n = 3/group). Values of p are shown numerically with p < 0.05 (independent samples t test). BMSC = bone marrow stromal cell; DMSO = dimethyl sulfoxide.

Fig. 3

mRNA targets of miR‐19a‐3p. (A) To assess transfection efficiency of miRNA‐sized nucleic acids, CalOBs were transfected with a nonlabeled control or an Alexa Fluor Red‐labeled fluorescent siRNA (same size as miRNAs) and visualized using fluorescence microscopy (555 nm). (B) CalOBs were transfected with negative control or miR‐19a‐3p miRNA mimics and expression measured by RT‐qPCR at 48 hours after transfection. (C) Volcano plot of RNAseq data highlighting selected genes that change with miR‐19a‐3p overexpression compared to control (p _adj < 0.05; log₂ FC > [1]). GSEA was performed on RNAseq data and showed enrichment for genes associated with (D) cellular senescence and (E) TP53 activity. (F) KEGG pathway analysis of significantly expressed genes shows regulation of cellular senescence pathway by miR‐19a‐3p. (G) Schematic highlighting some key players in cellular senescence pathway whose gene expression was significantly altered by miR‐19a‐3p. Chronic DNA damage ultimately results in increased p16 ^Ink4a and p21 ^Cip1 activity and subsequently decreased cell proliferation. This is accompanied by production of proinflammatory SASP driven by Janus kinase (JAK) signal transducer and activator of transcription (STAT) pathway that spreads senescence to neighboring healthy cells. The figure depicts the effects of miR‐19a‐3p overexpression on each of these genes using RNAseq data. RT‐qPCR was used to assess p16 ^Ink4a gene expression. Values of p are shown numerically with p < 0.05 (independent samples t test). CalOBs = calvarial osteoblasts; FC = fold‐change; GSEA = Gene Set Enrichment Analysis; SASP = senescence‐associated secretory phenotype.

Fig. 4

Effects of miR‐19a‐3p on p16 ^Ink4a and p21 ^Cip1 gene expression and proliferation. RT‐qPCR analysis of (A) miR‐19a‐3p, (B) p21 ^Cip1 , and (C) p16 ^Ink4a in control (NC mimic) and miR‐19a‐3p mimic transfected cells. Proliferative capacity of CalOBs transfected with control or miR‐19a‐3p miRNA mimics (n = 3/group) as shown by (D) cell proliferation assay, (E) crystal violet stain, and (F) percentage cell viability. Values of p are shown numerically with p < 0.05 (independent samples t test). CalOBs = calvarial osteoblasts; NC = negative control.

Fig. 5

Senotherapeutic effects of miR‐19a‐3p. (A) Calvarial osteoblasts were transfected with control (NC mimic) and miR‐19a‐3p mimic for 48 hours, followed by senescence induction using H₂O₂ in groups indicated in schematic diagram (designed using Biorender.com). RT‐qPCR analysis of p16 ^Ink4a and p21 ^Cip1 in (B) NC mimic, NC mimic + H₂O₂, and miR‐19a mimic + H₂O₂ transfected and treated cells (n = 3/group). RT‐qPCR analysis of Ki67 in (C) NC mimic, NC mimic + H₂O₂, and miR‐19a mimic transfected and treated cells (n = 3/group). Representative images of SA‐β‐Gal‐stained cells, (D) NC mimic, (E) NC mimic + H₂O₂, and (F) miR‐19a mimic + H₂O₂ (n = 3/group; magnification ×10). Values of p are shown numerically with p < 0.05 (independent samples t test). NC = negative control.