Aging-related changes of miR-23b-3p expression in extracellular vesicles from mesenchymal stromal cells affect TGFBR3 signaling

Abstract

Extracellular vesicles (EVs), which carry biological mediators such as microRNAs (miRNAs) and cytokines, play a crucial role in regulating recipient tissues and are considered a key mechanism of action in cell therapy. However, it has been found that cellular aging in donor cells significantly affects the expression profiles of these mediators involved in intercellular communication. In this study, we identified miR-23b-3p as a regulator of stemness-associated genes through its targeting of TGFBR3, with its expression differing between mesenchymal stem cells (MSCs) from young and aged donors. In aged MSCs, hypermethylation of the human MIR23B promoter region led to its downregulation. Treatment with the demethylating agent valproic acid restored miR-23b-3p expression and facilitated its incorporation into EVs. Our findings indicate that aging in MSCs alters the miRNA composition of EVs, potentially disrupting intercellular communication and significantly impacting the therapeutic efficacy of cell transplantation. To address this issue, the induction of demethylation may represent a promising strategy to improve treatment outcomes of MSCs.

Fig. 1

Expressions of miR23b-3p in the MSCs and its EVs are suppressed by donor aging. (A) Differential expression of miRNAs in EVs produced by MSCs of young mice (Yg-MSC) and that of aged mice (Ag-MSCs) shown by miRNA array analysis. (B) Expression levels of mmu-miR-23b-3p in EVs produced by the Yg-MSCs and the Ag-MSCs, analyzed by RT-PCR. Asterisk indicates significant differences at p < 0.05 between the comparison groups. Each bar represents the mean ± standard deviation from three independent experiments. (C) Comparison of cytoplasmic expression of mmu-miR-23b-3p in the primary MSCs prepared from young and aged mice. Asterisk indicates significant differences at p < 0.05 between the comparison groups. Each bar represents the mean ± standard deviation from three independent experiments. (D) MSCs in bone marrow tissue of young mice and aged mice were extracted by FACS as CD45-/Ter119-/PDGFRα + /Sca1 + fractions and compared for mmu-miR-23b-3p expression. Asterisk indicates significant differences at p < 0.05 between the comparison groups. Each bar represents the mean ± standard deviation from three independent experiments. (E) Expression of hsa-miR-23b-3p in the primary MSCs prepared from young and aged donors.

Fig. 2

mmu/hsa-miR-23b-3p targets TGFBR3 in MSCs prepared from aged donors. (A) Transfection of mmu-miR-23b-3p mimic RNA into the MSCs and changes in Tgfbr3 gene expression. Left: Changes in Tgfbr3 expression resulting from the transfection of the mimic RNA in the Ag-MSCs. Right: Changes induced by transfection of the mimic RNA in MSCs from aged donors. C. elegans miRNA cel-67-3p was used as a control. Different letters indicate significant differences were observed at P < 0.05. Each bar represents the mean ± standard deviation from three independent experiments. (B) Tgfbr3 expression after addition of Yg-MSC-derived EVs to Ag-MSCs. Asterisk indicates significant differences at p < 0.05 between the comparison groups. Each bar represents the mean ± standard deviation from three independent experiments. (C) Elevated expression of Tgfbr3 observed in the CD45-/Ter119-/PDGFRα + /Sca1 + MSC fraction isolated from aged mouse bone marrow. Asterisk indicates significant differences at p < 0.05 between the comparison groups. Each bar represents the mean ± standard deviation from three independent experiments. (D) Increased Tgfbr3 expression found in primary MSCs derived from aged mice. Asterisk indicates significant differences at p < 0.05 between the comparison groups. Each bar represents the mean ± standard deviation from three independent experiments.

Fig. 3

Tgfbr3 regulates genes involved in the proliferation, migration, and stemness of MSCs. (A) List of the top 15 proteins upregulated by transfection of mmu-miR-23b-3p mimic RNA into the Ag-MSCs. Asterisk indicates significant differences at p < 0.05. Each bar represents the mean ± standard deviation from three independent experiments. (B) Increased expression of the proliferation, metastasis, and stemness-related genes in the Ag-MSCs transfected with siRNA targeting Tgfbr3 (siTgfbr3). Asterisks indicate significant differences at p < 0.05. Each bar represents the mean ± standard deviation from three independent experiments.

Fig. 4

Upregulation of hsa-miR-23b-3p by demethylation in the MSCs prepared from aged donors. (A) Bisulfite qPCR analysis of the promoter region of the human MIR23B gene. Primers were specifically designed to target demethylated CpG sequences, such that PCR amplification occurs only when the sequence is demethylated. The Y-axis represents the Ct value, with higher Ct values indicating a lower degree of demethylation. Dots above the bars represent Ct values from individual donors. Asterisk indicates significant differences at p < 0.05 between the comparison groups. (B) Elevated expression of hsa-miR-23b-3p and ELOVL2 resulting from VPA + FGF2 treatment on MSCs from elderly donors. Asterisk indicates significant differences at p < 0.05. Each bar represents the mean ± standard deviation from three independent experiments. (C) Increased hsa-miR-23b-3p content detected in EVs isolated from human MSCs treated with the demethylation treatment. Asterisk indicates significant differences at p < 0.05. Each bar represents the mean ± standard deviation from three independent experiments.