miR-203-3p promotes senescence of mouse bone marrow mesenchymal stem cells via downregulation of Pbk

Abstract

The senescence of bone marrow mesenchymal stem cells (BMSCs) contributes to the development of degenerative skeletal conditions. To date, the molecular mechanism resulting in BMSC senescence has not been fully understood. In this study, we identified a small non-coding RNA, miR-203-3p, the expression of which was elevated in BMSCs from aged mice. On the other hand, overexpression of miR-203-3p in BMSCs from young mice reduced cell growth and enhanced their senescence. Mechanistically, PDZ-linked kinase (PBK) is predicted to be the target of miR-203-3p. The binding of miR-203-3p to Pbk mRNA could decrease its expression, which in turn inhibited the ubiquitination-mediated degradation of p53. Furthermore, the intravitreal injection of miR-203-3p-inhibitor into the bone marrow cavity of aged mice attenuated BMSC senescence and osteoporosis in aged mice. Collectively, these findings suggest that targeting miR-203-3p to delay BMSC senescence could be a potential therapeutic strategy to alleviate age-related osteoporosis.

Keywords: PDZ‐binding kinase; aging; bone marrow mesenchymal stem cells; miR‐203‐3p; osteoporosis.

FIGURE 1

Aged BMSCs exhibited increased level of cellular senescence. (a) FACS analysis of BMSC surface markers CD29, CD44, CD34, CD45, and HLA‐DR. (b) CCK8 assay for cell viability. (c) Edu assay for cell proliferation and quantitative analysis of positive cells, Bar: 100 μm. (d) Flow cytometry for cell cycle of both cell groups and quantitative analysis of cycle distribution. (e) Both osteogenic differentiation and adipogenic differentiation in 3W and 60W BMSCs, determined by Alizarin red staining and Oil red staining with quantification of osteogenic and adipogenic efficiency, Bar: 200 μm. (f) SA‐β Gal assay to determine cellular senescence and quantitative analysis of positive cells, Bar: 100 μm. (g) Heatmap of mRNA expression of phenotype‐related genes for cellular senescence analysis. (h) γ‐H2ax immunofluorescence to detect cellular DNA damage and quantitative analysis of fluorescence intensity, Bar: 5 μm. (i, j) Cellular expression of p16, p21 and p53 mRNA and protein expression levels and quantitative analysis, β‐actin served as loading control. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 2

High expression of miR‐203‐3p in BMSCs leads to reduced cell growth and promotes senescence. Overexpression of miR‐203‐3p in 3W‐BMSCs and silencing of miR‐203‐3p in 60W‐BMSCs were assayed in the corresponding two groups of cells, respectively. (a1, a2) CCK8 assay for cell viability. (b1, b2) Edu assay for cell proliferation and quantitative analysis of positive cells, Bar: 100 μm. (c1, c2) Flow cytometry was performed to detect the cell cycle of both groups of cells and to quantify the cycle distribution. (d1, d2, e1, e2) SA‐β‐gal assay to determine cellular senescence and quantify positive cells, Bar: 100 μm. (f1, f2) Heatmap of mRNA expression of genes related to cellular senescence analysis phenotype. (g1, g2) γ‐H2ax immunofluorescence assay to detect cellular DNA damage and quantify the fluorescence intensity, Bar: 5 μm. (h1, h2, i1, i2) Expression levels and quantitative analysis of p16, p21, p53 mRNA, and protein in both groups of cells. β‐Actin was used as a loading control. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 3

Pbk is a target of miR‐203‐3p. (a) Venn diagram of miR‐203‐3p target genes, overlapped according to seven predicted URLs (miRanda, MicroCosm, DIANA‐microT, ELMMo, PITA, PicTar and miRDB). (b) KEGG pathway of miR‐203‐3p target gene, mainly enriched in “M Phase, Mitotic Prophase.” (c) Relative expression of mRNA and protein of Pbk in young BMSCs and aged BMSCs as indicated. (d) Relative expression of mRNA and protein of the indicated Pbk after overexpression of miR‐203‐3p in young BMSCs. (e) Relative expression of mRNA and protein of the indicated Pbk after silencing of miR‐203‐3p in aged BMSCs. (f, g) Dual luciferase reporter gene analysis indicates that miR‐203‐3p can bind to the 3′‐UTR of Pbk. **p < 0.01; ***p < 0.001.

FIGURE 4

Pbk positively regulates the function of BMSC. (a1, a2, b1, b2) Pbk silencing in young BMSCs and Pbk overexpression in aged BMSCs. mRNA and protein expression levels were examined in the corresponding two groups of cells to confirm knockdown and overexpression efficiency, respectively. (c1, c2) Edu assay to detect cell proliferation and quantify positive cells, Bar: 100 μm. (d1, d2) Flow cytometry to detect cell cycle in both groups of cells and quantify cycle distribution. (e1, e2) SA‐β‐gal assay to determine cell senescence and quantify positive cells, Bar: 100 μm. (f1, f2) Heatmap of mRNA expression of phenotypically related genes for cell senescence analysis. (g1, g2) γ‐H2ax immunofluorescence to detect cellular DNA damage and quantify fluorescence intensity, Bar: 5 μm. (h1, h2, i1, i2) p16, p21, p53 mRNA and protein expression levels, and quantification in both groups of cells. β‐Actin was used as a loading control. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 5

Pbk promotes the degradation of p53. (a) The expression of Pbk and p53 was determined by protein blotting in young BMSCs after knockdown of the Pbk group and subsequent p53 treatment. (b) The rates of SA‐β‐gal‐positive cells in the four groups were compared and statistically analysed after knockdown of the Pbk group and then p53 treatment in 3W‐BMSCs. (c) Immunoprecipitation analysis of HA‐pbk transfected 60W BMSCs by protein blotting with anti‐HA or anti‐p53 antibodies. (d–f) Cycloheximide treatment followed by protein blotting for p53 protein stability analysis. (g) Ubiquitination assay in BMSC with and without ectopic Pbk expression. Immunoprecipitation of control IgG and p53 and protein blotting using anti‐p53 and anti‐ubiquitin antibodies to detect ubiquitin proteins (left). Identical loading levels of proteins and global ubiquitination profiles are shown (right). β‐Actin was used as a loading control. *p < 0.05; **p < 0.01.

FIGURE 6

Inhibition of miR‐203‐3p expression delays osteoporosis in aged mice. (a, e, f, g) Representative micro‐CT coronal and sagittal views of the mouse fibula. CTAn was used to measure bone bulk density (BV/TV%), trabecular thickness (Tb.Th, mm) and trabecular number (Tb.N, mm⁻¹). (b) Representative H&E‐stained sections of mouse fibula, Bar: 5 μm. (c, h) Representative ALP‐stained sections of mouse fibula and statistical evaluation of ALP staining expression. (d) Schematic representation of the process of miR‐203‐3p AAV inhibitor injection into the bone marrow cavity of mice. *p < 0.05; **p < 0.01; ***p < 0.001; ^# p < 0.05.