MicroRNA profiling reveals age-dependent differential expression of nuclear factor κB and mitogen-activated protein kinase in adipose and bone marrow-derived human mesenchymal stem cells

Abstract

Introduction: Mesenchymal stem cells (MSCs) play a central role in mediating endogenous repair of cell and tissue damage. Biologic aging is a universal process that results in changes at the cellular and molecular levels. In the present study, the role of microRNA (miRNA) in age-induced molecular changes in MSCs derived from adipose tissue (ASCs) and bone marrow (BMSCs) from young and old human donors were investigated by using an unbiased genome-wide approach.

Methods: Human ASCs and BMSCs from young and old donors were cultured, and total RNA was isolated. The miRNA fraction was enriched and used to determine the expression profile of miRNA in young and old donor MSCs. Based on miRNA expression, differences in donor MSCs were further investigated by using differentiation assays, Western blot, immunocytochemistry, and bioinformatics.

Results: Biologic aging demonstrated reduced osteogenic and adipogenic potential in ASCs isolated from older donors, whereas cell size, complexity, and cell-surface markers remained intact with aging. Analysis of miRNA profiles revealed that small subsets of active miRNAs changed secondary to aging. Evaluation of miRNA showed significantly decreased levels of gene expression of inhibitory kappa B kinase (IκB), interleukin-1α, inducible nitric oxide synthase (iNOS), mitogen-activated protein kinase/p38, ERK1/2, c-fos, and c-jun in MSCs from older donors by both bioinformatics and Western blot analysis. Nuclear factor kappa B (NF-κB), myc, and interleukin-4 receptor mRNA levels were significantly elevated in aged cells from both the adipose and bone marrow depots. Immunocytochemistry showed nuclear localization in young donors, but a cytosolic predominance of phosphorylated NF-κB in ASCs from older donors. Western blot demonstrated significantly elevated levels of NF-κB subunits, p65 and p50, and AKT.

Conclusions: These findings suggest that differential expression of miRNA is an integral component of biologic aging in MSCs.

Figure 1

Characterization of age-dependent differences in adipose stem cells (ASCs) and bone marrow stem cells (BMSCs). (a) Representative photomicrographs of MSCs show undifferentiated control cells (left), lipid inclusions with Oil Red O (middle), and mineralization with Alizarin Red (right) in ASCs from young (top) and old (bottom) donors. Original magnification of undifferentiated and mineralized cultures was ×10, and lipid inclusions were at ×40. (b) Quantification of differentiation potentials of ASCs from young and old donors shown as OD normalized to protein for cells cultured in osteoblastogenic and adipocytogenic conditions. Bars represent mean ± SEM and are representative of three quantifications. *P < 0.05. (c) A representative plot from flow cytometry shows forward versus side light scatter used to assess cell size and heterogeneity of MSCs. Green plots demonstrate ASCs, and red plots indicate BMSCs. Data for young donors are on the right, and for old donors, on the left.

Figure 2

Heatmap and hierarchic clustering of miRNA expression in adipose stem cells (ASCs) and bone marrow stem cells (BMSCs). (a) Expression of miRNA in old and young BMSC donors, and mapping of hierarchic clustering of miRNA and heatmap display of miRNA profiles. (b) Expression of miRNA in old and young ASC donors, and mapping of hierarchic clustering of miRNA and heatmap display of miRNA profiles. Columns represent individual donor samples, and each row represents individual assayed miRNA. Green represents downregulation of miRNAs, and red represents upregulation of miRNAs.

Figure 3

Age-dependent changes in miRNA profiles of adipose stem cells (ASCs) and bone marrow stem cells (BMSCs). (a) Statistically significant up- and downregulated miRNA in BMSCs. (b) Statistically significant up- and downregulated miRNA in ASCs. (c, d) Volcano plot of miRNA exhibiting P < 0.05 in expression as a result of BMSCs donor age. (e, f) Volcano plot of miRNA exhibiting P < 0.05 in expression as a result of ASCs donor age. (g) Fold regulation of significant miRNA in old versus young BMSCs donors. (h) Fold regulation of significant miRNA in old versus young ASCs donors. Upregulated miRNA are denoted in red, downregulated miRNAs are green, and miRNAs not statistically significant are black. Horizontal blue line represents P-value cutoff (P < 0.05), and vertical grey lines represent fold-change cutoff (more than twofold).

Figure 4

Canonic pathway of predicted gene expression influenced by miRNA in adipose stem cells (ASCs) and bone marrow stem cells (BMSCs). Canonic pathways derived from predicted putative targets of miRNA action on gene expression by using Ingenuity Pathways Assessment (IPA) analysis of MSCs from old versus young donors. (a) Downregulated miRNA in BMSCs from old compared with young donors. (b) Upregulated miRNA in BMSCs from old, compared with young donors. (c) Downregulated miRNA in ASCs from old, compared with young donors. (d) Upregulated miRNA in ASCs from old, compared with young donors. Yellow line represents P-value cutoff (P < 0.05), and yellow dots represent ratio of projected involvement of targets to actual inputted involvement. Height of bars is determined by projected involvement of particular pathway.

Figure 5

Predicted biologic functions influenced by miRNA expression in adipose stem cells (ASCs) and bone marrow stem cells (BMSCs). Major biologic functions influenced by miRNA action on gene expression are shown from Ingenuity Pathways Assessment (IPA) analysis of MSCs from old and young donors. Involvement of biologic functions is determined from network and focus molecule association with miRNA targets in IPA. (a) Downregulated miRNA in BMSCs from old donors, compared with young donors. (b) Upregulated miRNA in M-MSCs from old donors, compared with young donors. (c) Downregulated miRNA in ASCs from old donors, compared with young donors. (d) Upregulated miRNA in ASCs from old donors, compared with young donors. The yellow line represents P-value cutoff (P < 0.05).

Figure 6

Top network analysis of focus molecules by Ingenuity Pathways Assessment (IPA) in bone marrow stem cells (BMSCs). (a, b) Top two networks of focus molecules generated by IPA in direct or indirect regulation of gene expression by downregulated miRNA in BMSCs. (c, d) Top two networks of focus molecules generated by IPA of directly and indirectly involved gene-expression regulation by upregulated miRNA in BMSCs. Gray symbols, directly involved molecules, those inputted into IPA; white symbols, indirectly involved molecules, those commonly associated with the genes/pathways demonstrated.

Figure 7

Top network analysis of focus molecules by Ingenuity Pathways Assessment (IPA) in adipose stem cells (ASCs). (a, b) Top two networks of focus molecules generated by IPA in direct or indirect regulation of gene expression by downregulated miRNA in ASCs. (c, d) Top two networks of focus molecules generated by IPA of directly and indirectly involved gene-expression regulation by upregulated miRNA in ASCs. Gray symbols, directly involved molecules, those inputted into IPA; white symbols, indirectly involved molecules, those commonly associated with the genes/pathways demonstrated.

Figure 8

Evaluation of miRNA influence on mRNA levels in adipose stem cells (ASCs) from old and young donors. (a) The plot demonstrates significant up- and downregulated mRNA in ACSs from old versus young donors. (b) Fold regulation of significant mRNA assessed in ACSs from old versus young donors. (c) Heatmap shows old versus young donor mRNA expression levels; green represents downregulation of mRNAs, and red represents upregulation of mRNAs. (d) The plot shows the top canonic pathways generated from Ingenuity Pathways Assessment (IPA) analysis of mRNA differences in ACSs from old versus young donors. Yellow line, P-value cutoff (P < 0.05), and yellow dots, ratio of projected involvement of targets to actual inputted involvement. Height of bars is determined by projected involvement of particular pathway. (e) The plot shows top predicted biologic functions from the mRNA data inputted into IPA. Yellow line, P-value cutoff (P < 0.05). (f through i) The top four networks involved in mRNA differences between ACSs from old versus young donors. White items, indirectly involved molecules, those commonly associated with the genes/pathways demonstrated. Green items, downregulated molecules directly entered into IPA. Red items, upregulated molecules based on fold change of mRNA.

Figure 9

Western blot analysis of differences in protein levels resulting from donor age and miRNA influences. (a) Immunocytochemical localization of p65 and p50 subunits of NF-κB in representative ASCs from old and young donors. Images are at ×20 magnification. Negative controls are representative images per age group. Images are at ×5 magnification. (b) Representative Western blots of NF-κB pathway elements in ASCs from young (Y) and old (O) donors. (c) Representative Western blots of MAPK/ERK pathway elements in ASCs from young (Y) and old (O) donors. (d) The quantitative fold change in expression of NF-κB pathway proteins normalized to total actin levels. (e) The quantitative fold change in MAPK signaling expression of proteins normalized to total actin levels. Bars, mean ± SEM of at least three Western blot quantifications. *P < 0.05; **P < 0.01; ***P < 0.001).