MicroRNA-141-3p Negatively Modulates SDF-1 Expression in Age-Dependent Pathophysiology of Human and Murine Bone Marrow Stromal Cells

Abstract

Stromal cell-derived factor-1 (SDF-1 or CXCL12) is a cytokine secreted by cells including bone marrow stromal cells (BMSCs). SDF-1 plays a vital role in BMSC migration, survival, and differentiation. Our group previously reported the role of SDF-1 in osteogenic differentiation in vitro and bone formation in vivo; however, our understanding of the post-transcriptional regulatory mechanism of SDF-1 remains poor. MicroRNAs are small noncoding RNAs that post-transcriptionally regulate the messenger RNAs (mRNAs) of protein-coding genes. In this study, we aimed to investigate the impact of miR-141-3p on SDF-1 expression in BMSCs and its importance in the aging bone marrow (BM) microenvironment. Our data demonstrated that murine and human BMSCs expressed miR-141-3p that repressed SDF-1 gene expression at the functional level (luciferase reporter assay) by targeting the 3′-untranslated region of mRNA. We also found that transfection of miR-141-3p decreased osteogenic markers in human BMSCs. Our results demonstrate that miR-141-3p expression increases with age, while SDF-1 decreases in both the human and mouse BM niche. Taken together, these results support that miR-141-3p is a novel regulator of SDF-1 in bone cells and plays an important role in the age-dependent pathophysiology of murine and human BM niche.

Bone marrow stromal cells (BMSCs) are progenitor cells that can differentiate into numerous different cell types including osteoblasts, osteocytes, chondrocytes, and adipocytes (1). These cell types provide the cellular framework for bone, muscle, cartilage, tendon, and ligaments. The differentiation of BMSCs into their final cellular type is a highly synchronized and complex process. Differentiation of BMSCs involves several molecular factors, including growth factors and cytokines (2–7). Cytokines are well known for their role in embryogenesis, combating infection, and regulating inflammation. Recent studies have also shown that cytokines play a vital role in BMSCs’ differentiation (2). One example of a specific BMSC regulator is stromal cell-derived factor-1 (SDF-1), also known as CXCL12 (8).

SDF-1 is a chemokine protein that binds primarily to cysteine (C)-X-C chemokine motif receptor-4 (CXCR4) and plays an important role in the homeostasis of organ development and differentiation of hematopoietic cells (9,10). The interaction between SDF-1 and CXCR4 is known for controlling BMSCs migration, osteogenic differentiation, and cell survival from oxidative stress (11–17). SDF-1 has also proven to accelerate and enhance calcium deposition, regulate several osteogenic factors (runt-related transcription factor 2 or RUNX-2, BMP-2, osteocalcin, collagen alpha 1 type 1), enhance alkaline phosphatase activity, and maintain bone homeostasis by assisting in BMSC migration to distant sites of injury (18). Our group previously reported that SDF-1 plays a vital role in osteogenic differentiation in vitro and bone formation in vivo (18). Bone fracture healing has also been demonstrated to be potentiated by BMSCs and the SDF-1/CXCR4 interaction (19). Because of the clinical significance of SDF-1 in BMSCs’ differentiation and bone tissue engineering, it is critical to better understand the mechanism(s) by which SDF-1 is regulated. SDF-1 has been found to decline in specific tissue compartments with aging as well as other pathophysiological conditions (20,21). Decline in SDF-1 expression could be due to transcriptional regulation or may be due to translational inhibition. MicroRNAs are involved in post-transcriptional regulation of messenger RNA (mRNA) by RNA interference. MicroRNAs are small noncoding RNAs that target mRNA sequences by binding the 3′-untranslated regions (3′-UTRs), which causes mRNA degradation or block translation (22,23). Because of miRNA’s ability to inhibit mRNA translation, it is possible that a miRNA is involved in the regulation of SDF-1, subsequently affecting BMSC differentiation and osteogenesis. MicroRNA’s ability to regulate cell differentiation, cell proliferation, and osteogenesis is well established (24–26). Furthermore, a number of studies indicate that miRNAs are significantly altered with aging (27,28) and in the bone marrow (BM) microenvironment (29). Our bioinformatics analysis showed that a number of miRNAs target the 3′-UTR of SDF-1 including miR-141-3p. miR-141-3p has been found to be of particular interest because of its role in osteogenesis differentiation and oxidative stress. In this study, we investigated the post-transcriptional regulation of SDF-1 and miR-141-3p in mouse and human BMSCs.

Materials and Methods

BMSC Isolation and Expansion

Human BM from young (18–40 years of age) and old (60–85 years old) subjects (n = 10/group) was obtained under sterile conditions from orthopedic surgery patients as per Institutional Review Board (IRB) of Augusta University. All patients gave written informed consent. The CD271 positive (+) MSCs were isolated according to the manufacturer’s protocol by using a kit (Miltenyi Biotec Inc, 130-092-283). For aging studies, direct isolation procedure was used to quickly capture CD271+ MSCs from BM without culturing or standard plastic adherence. For other studies, CD271+ MSCs were isolated directly from BM; washed with standard culture medium composed of Dulbecco’s Modified Eagle Medium (DMEM) (Corning, 10-013-CM), 1% antibiotics–antimycotics (AA; Invitrogen, 15240-062), and 15% fetal bovine serum (FBS); transferred to 100 mm culture dish; and incubated at 37°C in humidified atmosphere at 5% carbon dioxide (CO₂). After 24 hours, the medium with nonadherent cells was removed, and the adherent cells carefully washed in Dulbecco’s phosphate-buffered saline (DPBS) and further expanded in fresh standard culture medium. Murine BM interstitial fluid and BMSCs (n = 10) were isolated from the long bones of C57BL/6 mice as previously described (30). For BM interstitial fluid collection, total BM aspirates were placed in Eppendorf centrifuge tubes followed by centrifugation for 10 minutes at 2,500 rpm at 4°C. The cell-free supernatant was collected in fresh Eppendorf tubes and stored at −80°C until use.

MicroRNA Mimic and Inhibitor Transfection in BMSC

Transient transfections of miRNA mimics and inhibitors were performed using HiPerFect transfection reagent (Qiagen, 301704, Valencia, CA) according to manufacturer’s protocol. Mimics and inhibitors of miR-141-3p were purchased from QIAGEN. Culture-expanded CD271+ MSCs were first seeded at 2 × 10⁴ cells/cm² in a 24-well plate followed by transfection of mimic/inhibitor (5 nM mimic or 50 nM inhibitor as final concentration) after 24 hours in 1% fetal bovine serum (FBS) culture media. The cell lysates and the conditioned medium were harvested subsequent to the different experimental conditions and used for miRNA and mRNA qPCR assays as well as enzyme-linked immunosorbent assay (ELISA).

Quantitative Real-Time PCR for RNA and miRNA

For RNA analysis, BMSCs were lysed by TRIzol. RNA isolation and subsequent cDNA synthesis (Bio-Rad, 170–8891) were performed as previously described (1,30). Quantitative real-time PCR amplification was performed according to procedures reported previously (1,30) with custom-designed qPCR primers (Table 1). The expression levels of mRNA were normalized to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin. Relative expression of mRNA was evaluated by using the comparative CT method (ΔΔCt).

Table 1.

Nucleotide Sequences of Mouse Primers Used for RT-PCR

Gene	Primer	Product Size in Base Pair	Reference/Accession Number
GAPDH (Mouse)	CAT GGC CTC CAA GGA GTA AGA	105	M32599
	GAG GGA GAT GCT CAG TGT TGG
SDF-1 (Mouse)	GTG AGA ACA TGC CTA GAT TTA CCC	105	(18)
	ATA GGA CTC AGG GAC AAT TAC CAA
SDF-1 (Human)	TAT TAC TGG GAC TGT GCT CAG AGA	110	NM_199168.3
	TGA TCA GGC TAC AGA AAT GAG AAG
BMP-2 (Human)	ATG TTA GGA TAA GCA GGT CTT TGC	81	NM_001200.2
	GAC CTT TTT CTC TTT TGT GGA GAG
RUNX-2 (Human)	GTA CCA GAT GGG ACT GTG GTT ACT	82	NM_001015051.3
	CTC AGA TCG TTG AAC CTT GCT ACT
C/EBP-a (Human)	TGG ACA AGA ACA GCA ACG AGT A	109	NM_004364.3
	CCA CGA CCT AGC TTT CTG GT
β-actin (Human)	ACA TGT ATG AAG GCT TTT GGT CTC	96	NM_001101.3
	GTG TGC ACT TTT ATT CAA CTG GTC

Note: Abbreviations: RT-PCR = Reverse transcription polymerase chain reaction; SDF-1 = Stromal cell-derived factor-1.

For miR-141-3p quantitation, miRNA was isolated from cells using the miRNA easy isolation kit and reverse transcribed into cDNA using miScript II RT kit (Qiagen, 218160). Fifty picograms of cDNA were amplified in each qRT-PCR using SYBR Green I and miR-141-3p-specific primers (Qiagen). The average of RNU6 (RNA, U6 small nuclear 2) and SNORD (small nucleolar RNA, C/D box) was used as normalization reference genes for miRNA.

Transwell Migration Assay

In vitro chemotaxis was assayed using the HTS Transwell 96-well system (8 μm pore size; Corning, 3374 and 3583) as previously described (31). In brief, transwell migration of murine BMSCs (from 3-month-old mice) was used to assess the bioactivity of BM interstitial fluid SDF-1 from young (3 months old) and old (18 months old) mice (n = 10/group) placed in the lower chambers as an assay for SDF-1 bioactivity. For human cells, transwell migration from a young subject (18 years old) was assessed with BM interstitial fluid from young (18–40 years of age) and old (60–85 years old) human subjects (n = 10/group) in the lower chambers for SDF-1 bioactivity. The difference in migration between BMSCs placed in the upper chamber and treated with either the CXCR4 inhibitor AMD3100 (400 µg/ml), or saline, shows the migration mediated by SDF-1 activation of CXCR4.

SDF-1 ELISA

SDF-1α protein levels were measured by enzyme-linked immunosorbent assay (ELISA) (R&D Systems human or murine SDF-1α DuoKits) either in BM aspirate supernatants or cell culture medium as described earlier (18). Each sample was tested in duplicate. Standard curves were reconstituted using human or murine SDF-1α standards (R&D Systems). The optical density of each well was determined using a microplate reader set to 450 nm wavelength. SDF-1α protein concentration was calculated using standard curves.

Luciferase Reporter Assay

Putative miRNA-binding sites on the 3′-UTR of SDF1 mRNA were identified using TargetScan online software. Custom reporter vectors were purchased from Genecopoeia created by cloning the wild-type or mutant 3′-UTR of SDF1 mRNA (accession number NM_000609.6) into the pEZX-MT06 with dual firefly and Renilla luciferase reporters. Firefly and Renilla luciferase activities were analyzed using the Dual-Luciferase reporter assay system (Genecopoeia, LF001) according to the manufacturer’s protocol. In brief, human BMSCs were seeded at 1.5 × 10⁴ cells/cm² in a 96-well plate (Costar, 3917) and cotransfected with the 3′-UTR (SDF1-3′-UTR) reporter plasmids together with miR-141-3p mimics or control siRNA using DharmaFECT DUo transfection reagent (Fisher Thermo Scientific, 2010-01). Luciferase activity was analyzed 24 hours post-transfection using Luciferase Assay reagents (Genecopoeia, LF001).

Statistical Analysis

GraphPad Prism 5 (La Jolla, CA) was utilized to perform analysis of variance with Bonferroni pair-wise comparison or unpaired t-tests as appropriate. A p value of <.05 was considered significant.

Results

Decline in SDF-1 Level With Age in Human and Mouse BM Interstitial Fluid Levels

With aging, there is an increase in oxidative stress in organs and tissues of body, which affects the normal physiological levels of hormones and cytokines. In this study, we investigated the levels of SDF-1 in BM interstitial fluid. Our data demonstrated that there was a significant decline in SDF-1 levels in BM interstitial fluid with age in both mice (p value = .01) and humans (p value = .01; Figure 1). We also noted that this cytokine had the ability to induce AMD3100 sensitive BMSC migration, a marker of SDF-1 bioactivity that reflects the summation of SDF-1 expression and inactivation (Figure 2). For murine BM interstitial fluid, the majority of migration is mediated by AMD3100 sensitive SDF-1 bioactivity (p < .001). This was also true for young human BM interstitial fluid (p < .001). For the old human BM interstitial fluid, there is little or no bioactive SDF-1. This result suggests that there is a decrease both in SDF-1 protein levels and in the bioactivity of the remaining SDF-1 with age in the BM niche (Figures 1 and 2)

Figure 1.

SDF-1 concentrations decrease with age in (a) murine and (b) human BMISF. Mean values ± SD for n = 10 per age group. For murine, BMISF was collected from young (3 months of age) and old (22 months of age) mice and normalized to protein. For humans, BMISF was obtained from young (18–40 years of age) and old (60–85 years old) subjects (n = 10/group, * p < .05, ****p < .0001). Abbreviations: BMISF = Bone marrow interstitial fluid; SDF-1 = Stromal cell-derived factor-1.

Figure 2.

SDF-1 bioactivity in murine and human BMISF declines with age. Transwell migration assay of (a) murine BMSCs (from 3-month-old mice) was used to assess the bioactivity of BMISF SDF-1 from young (3 months old) and old (18 months old) mice (n = 10/group) placed in the lower chambers of transwell plate for SDF-1 bioactivity and (b) human BMSCs from a young subject (18 years old) were assessed with BMISF from young (18–40 years of age) and old (60–85 years old) human subjects (n = 10/group) in the lower chambers of transwell plate for SDF-1 bioactivity. *p < .01, **p < .001, ***p < .0001 relative to young non-AMD3100-treated controls (a) or, old non-AMD3100-treated controls (b) samples. Abbreviations: BMISF = Bone marrow interstitial fluid; BMSCs = Bone marrow stromal cells; SDF-1 = Stromal cell-derived factor-1.

SDF-1 Is a Target of miRNA-141-3p

MicroRNAs are small RNA molecules that play a major role in the negative regulation of genes. To determine the novel mechanism of SDF-1 regulation in BMSCs, we used bioinformatics tools (targetScan) to identify the miRNAs targeting SDF1. We identified several miRNAs, including miR-141-3p, targeting SDF1 (Figure 4). Furthermore, the binding sites for miRNA-141-3p at the 3′-UTR of SDF-1 are homologous throughout primates and mammals (Figure 4). We were most interested in miR-141-3p because of its role in osteogenic differentiation. Previously, we demonstrated the negative role of miR-141-3p in BMSCs’ differentiation (1). We hypothesized that miR-141-3p targets SDF-1 and inhibits its expression in BMSCs. Initial experiments showed that transfection of miRNA-141-3p mimic in mouse and human BMSCs significantly (p value = .05) inhibits SDF-1 expression (Figure 3). To further verify these results, functional assays were performed using luciferase reporter assays. The luciferase plasmid constructs containing putative 3′-UTR sequence of SDF-1 were used. Cotransfection of miRNA-141-3p mimic in human BMSCs showed significantly (p value = .01) suppressed luciferase reporter activity, indicating that miR-141-3p suppresses SDF-1 expression (Figure 4). Bioinformatics analysis showed miR-141-3p binds SDF1 3′-UTR at two sites, first at 51–58 and second at 2940–2947 nucleotide position (Figure 4a). Mutation of either of the two binding sites did not abolish the inhibitory effect of miR-141-3p, but mutation of both miR-141-3p-binding sites completely eliminated the inhibitory effects (Figure 4b). These data demonstrate that SDF1 is indeed the direct target of miR-141-3p.

Figure 4.

Luciferase reporter assays demonstrating the SDF1 3′-UTR is the target of miR-141-3p. (a) Schematic representation of the 3′-UTR of human SDF1 mRNA with the two putative complementary sequences to miR-141 and interspecies conservation of putative miR-141 binding sites within the SDF1 (b) Luciferase activity assays detecting the miR-141-3p binding sites on the 3′-UTR of SDF1. Cotransfection of control miRNA/siRNA and luciferase reporters with the wild-type SDF1 3′-UTR was used as a control. In other groups, miR-141-3p mimics were cotransfected with luciferase reporters containing wild-type 3′-UTR or the 3′-UTR with mutated binding site 1 (M1/positions 51–58), with mutated binding site 2 (M2/positions 2940–2947), and with both mutations (M3). The control mimic was independently set to 100%. Data are presented as mean ± SEM (n = 5; #p < .01). Abbreviations: 3′-UTR = 3′-untranslated region; SDF-1 = Stromal cell-derived factor-1.

Figure 3.

miR-141-3p reduces SDF1 in mice and human BMSCs: (a) Mouse and (b) human BMSCs were transfected with miRNA mimic miR-141 and NC control (nontargeting siRNAs) for 48 hours. These cells were then used for RNA isolation. Data (means ± SD, n = 5) are represented as the fold change in expression compared with control (*p < .05, #p < .01). Abbreviations: BMSCs = Bone marrow stromal cells; SDF-1 = Stromal cell-derived factor-1.

miRNA-141-3p Regulates Bone-Related Genes in Human BMSCs

Our group previously reported that miRNA-141 inhibits osteogenic differentiation of mouse BMSCS in the presence of osteogenic culture conditions (1). We hypothesized that miR-141-3p might play a similar role in human BMSCs’ biology. The undifferentiated human BMSCs were transfected with the miR-141-3p mimetic and negative control (control) for 48 hours followed by real-time PCR on BMP-2, RUNX2, and C/BEPa2. Real-time PCR data showed significant downregulation of osteogenic genes. The BMP-2 (p value = .01) and RUNX2 (p value = .05) were significantly upregulated and C/BEPa2 was significantly (p value = .05) downregulated compared with control groups (Figure 5). Our data indicate that miR-141-3p plays a vital role in human BMSCs gene regulation.

Figure 5.

miR-141-3p regulates bone-related genes in human BMSCs: Human BMSCs transfected with miR-141-3p and nontargeting control (NC control) mimics for 48 hours followed by real-time PCR on (a) BMP-2, (b) RUNX2, and (c) C/BEPa2. Data (means ± SD, n = 5) are represented as the fold change in expression compared with control (*p < .05, #p < .01). Abbreviation: BMSCs = Bone marrow stromal cells.

Dysregulation of miR-141-3p in the Aged BM Microenvironment

The BM microenvironment is important for the normal health of bone, musculoskeletal, and body defense mechanisms. Our initial experiments showed that SDF-1 is downregulated in aged BM interstitial fluid (Figure 1). We speculated that miRNA-141-3p targeting SDF-1 might play an important role in the decline in the level of SDF-1. We therefore measured miRNA-141 levels in the BM microenvironment. We found that miR-141-3p miRNA was significantly (p value = .03) upregulated in mouse BM interstitial fluid. We also noted a trend (p value = .09) of upregulation of miRNA-141-3p in aged human BMSCs (Figure 6). These findings suggest that miRNA-141-3p might play a key role in the decline in SDF-1 levels in the aged BM microenvironment.

Figure 6.

Age-related dysregulation of miR-141-3p on mouse and human bone marrow niche. Real-time PCR of miRNA-141-3p on (a) young (n = 6) and old (n = 5) mouse bone marrow interstitial fluid and (b) young age 18–40 (n = 9) and old age 60 and above (n = 11) human bone marrow stromal cells. Data (means ± SD) are represented as the fold change in expression compared with young (*p value = .05).

Discussion

SDF-1 is a cytokine that plays a vital role in BMSC migration and osteogenic differentiation (10). Our group and others have demonstrated that SDF-1 has an active role in bone formation, cell survival, and fracture healing (11–19). Aging is an important risk factor in musculoskeletal degeneration (32–34). In this study, we analyzed SDF-1 expression in the aged human and mouse BM niche because of its vital role in bone health, repair, and regeneration. Our data demonstrated that SDF-1 expression declines with age in the BM environment of both humans and mice. Previously, Loh and colleagues (20) reported that SDF-1 alpha expression levels decreased in aged wounds and significantly impaired wound healing, reduced granulation tissue, and increased the epithelial gap. Consequently, understanding how SDF-1 is regulated in the BM niche during pathophysiological conditions is important to develop BMSC-dependent therapeutic approaches. Previous studies have shown that SDF-1 is regulated by several transcriptional factors (35–37).

In this study, we focused on the regulation of SDF-1 expression at a post-transcriptional level. Post-transcriptional regulation of gene expression can be controlled by microRNAs (22,23). Increasing data indicates the vital role of miRNAs in regulating BMSC osteogenic differentiation (24,25,38) and bone pathophysiology (1,24–26). MicroRNA accomplishes this through post-transcriptional changes via binding the 3′-UTR and interfering with mRNA translation and/or mRNA stability (22,23). We utilized bioinformatics analysis to predict the miRNA that would bind to the 3′-UTR of SDF-1. We identified miRNA-141-3p targets SDF-1 via two binding sites on the 3′-UTR of SDF-1. Moreover, the novel binding sites for miRNA-141-3p at the 3′-UTR of SDF-1 are homologous throughout mice, humans, chimpanzees, rhesus, and rats. MicroRNA-141-3p is a part of the miRNA 200 family, which includes miRNAs 141, 200a, 200b, 200c, and 429; miRNA-141 is found on chromosome 12 in humans and on chromosome 6 in mice (39). Preliminary studies had shown that transfection with the miR-141-3p mimetics decreases SDF-1 expression in mouse BMSCs. We further verified that SDF-1 is a target of miR-141-3p using a luciferase reporter assay. It has been previously reported that SDF-1 is the target of several other miRNAs. For example, Dong and colleagues (40) observed that miRNA-137 acts as a tumor suppressor by targeting CXCL12, and Fan and colleagues (41) found that miRNA-454 regulates SDF-1 in pancreatic ductal adenocarcinomas; however, we are the first group to identify miRNA-141 as a key regulator of CXCL12 (SDF-1) expression in BMSCs. Furthermore, we tested the role of miRNA-141 and its impact on osteogenesis in human BMSCs. Our data demonstrated that human BMSCs treated with miRNA-141-3p decreased BMP-2 and RUNX-2 expression and increased C/BEPa2. These findings reveal that miRNA-141-3p downregulates the expression of SDF-1 in human and mouse BMSCs, as well as osteogenic pathway molecules downstream of SDF-1 signaling (BMP-2 and RUNX-2).

BMSCs are pluripotent mesenchymal stem cells that can differentiate into numerous cell types including osteoblasts, adipocytes, chondroblasts, and myoblasts (42,43). SDF-1 is known to play a critical role in BMP-2- and RUNX-2-dependent osteogenesis, bone remodeling, and bone healing (18,44,45). We believe that our data showing decreased levels of SDF-1, BMP-2, and RUNX-2 in the presence of miRNA-141-3p mimetics suggest that this miRNA has antiosteogenic properties. While our data demonstrate the ability of miRNA-141-3p to suppress osteogenic differentiation through its interaction with SDF-1, it is likely that other factors are also in play. MicroRNAs typically target more than one gene, which makes them challenging to study and identify their precise role in cell signaling. The effects of miRNA-141-3p on BMSC differentiation into osteogenic cells may involve targeting other genes in addition to SDF-1. Previously, we demonstrated that miR-141-3p repressed sodium-dependent vitamin C transporter 2 expression at the functional level by targeting the 3′-UTR of mRNA (1). We also demonstrated that miR-141 decreased osteogenic differentiation in mouse BMSCs (1). Furthermore, Itoh and colleagues (46) reported that miRNAs-141 and 200a target Dlx5 to inhibit bone formation. It is probable that miRNA-141’s negative effect on bone formation is the result of multiple gene targets. Interestingly, we also found that the aged BM environment displays higher levels of miRNA-141-3p and decreased levels of SDF-1 in both human and mouse BMSCs. Elevated microRNA-141-3p and lower SDF-1 could therefore contribute to the pathophysiology of BMSCs that is noted to occur with aging. The elevated levels of miR-141 in the aged BM niche may be due to an increase in oxidative stress with age. Oxidative stress is an important factor in the pathophysiology of aging. It has been previously reported that miR-141 is elevated in the presence of oxidative stress in various human and mouse cell types (47–49). We also noted that increased oxidative stress on mouse BMSCs decreases SDF1 level (data not shown). Our group previously reported that SDF1 protected murine BMSCs from H₂O₂-induced cell death through increasing autophagy and decreasing apoptosis in mouse BMSCs in vitro (14). We speculate that oxidative stress elevates miR-141-3p gene expression, which is directly associated with decrease in SDF-1 level. The antagomirs of miR-141-3p might elevate SDF1 protein level and reverse this effect.

In conclusion, our study is the first to report a relationship demonstrating the microRNA-141-3p and SDF-1 signaling axis linkage in age-dependent pathophysiology of human and mice BMSCs. Our study demonstrated that elevated level of miRNA-141-3p and decline in SDF1 level could have a significant negative impact on bone and BM environment with aging. Inhibitors (antagomirs) of miR-141-3p or SDF1 overexpression could potentially be developed as therapeutic agents to address age-related bone pathophysiology. These findings are critical in understanding the precise mechanisms of age-dependent osteogenic differentiation and musculoskeletal pathophysiology.

Funding

This publication is based upon work supported in part by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research, and Development Program (VA Merit Award 1I01CX000930 01, W.D.H.) and the National Institutes of Health (National Institute on Aging-AG036675 MH, CS, W.D.H.). The contents of this publication do not represent the views of the Department of Veterans Affairs or the U.S. Government. The above-mentioned funding did not lead to any conflict of interests regarding the publication of this manuscript. The authors also declare that there is no other conflict of interest regarding the publication of this manuscript.

Conflict of Interest

None reported.