Abstract
Purpose
Osteoporosis (OP) is influenced by dysregulated miRNAs, particularly during osteoblast differentiation. The precise mechanisms are still under debate. This study aimed to explore the impact of bone marrow mesenchymal stem cells (BMSCs)-derived exosomal miR-133b-3p on the TGF-β1/Treg-mediated immune pathway, offering insights into OP’s pathogenesis and potential therapeutic targets.
Materials and methods
Bioinformatics analysis of GEO dataset (GSE64433) identified differentially expressed miRNAs in osteoporosis. Target genes were predicted using TargetScan, miRDB, miRTarBase, and miRWalk databases, followed by GO and KEGG pathway enrichment analyses. An OP rat model was constructed by ovariectomy (n = 36, randomly allocated into three groups: control, OP, and OP+exosomal miR-133b-3p, n = 12 per group). BMSCs were isolated at 12 weeks post-OVX.Flow cytometry was used to identify the surface markers of BMSCs, CD29, CD44, CD106, CD34, and CD45. Exosomes were isolated from passages 3–5 BMSCs using ExoQuick kit. Transmission electron microscopy and nanoparticle tracking analysis were used to observe the morphology and size distribution of exosomes, and the expression of exosomal protein markers CD9, CD63, and TSG101 was detected by Western blot. qRT-PCR was performed to detect miR-133b-3p and TGF-β1 expression in exosomes. Dual-luciferase reporter assay validated the direct interaction between miR-133b-3p and TGF-β1 3’-UTR. Dual-energy X-ray bone densitometry was used to detect bone mineral density (BMD) after 4 weeks of treatment with miR-133b-3p-enriched exosomes (200 μg weekly via tail vein injection). Micro-CT was used to analyze the BV/TV, Tb.N, Tb.Th, SMI, Ct.Th, BA/TA, and Tb.Sp. In vitro experiments using isolated CD4+ T cells were conducted to assess TGF-β1 expression and CD4 + CD25 + Foxp3+ Treg cell differentiation via Western blot, RT-PCR, and flow cytometry. Osteoclast marker enzymes TRAP, MMP-9, and Cathepsin K were identified using immunohistochemistry.
Results
Bioinformatics analysis revealed 27 differentially expressed miRNAs. Target prediction of miR-133b-3p identified 44 high-confidence genes, with TGF-β1 emerging as a key target. BMSCs expressing CD29, CD44, and CD106 (but not CD34 and CD45) were isolated from both control and OP rats. The identified exosomes were roughly spherical with a double-layered membrane, they had a size distribution of about 103.5 ± 8.2 nm and 105.8 ± 10.6 nm, respectively, and had a positive expression of CD9 CD63, and TSG101. qRT-PCR analysis revealed significantly decreased miR-133b-3p expression in OP group exosomes (P < 0.001). Dual-luciferase assay confirmed direct binding of miR-133b-3p to TGF-β1 3’-UTR. Treating OP rats with exosomal miR-133b-3p improved various bone metrics, increased BV/TV, Tb.N, Tb.Th, BMD, Ct.Th, and BA/TA, decreased Tb.Sp and SMI, and improved bone histopathological changes in rat bone tissue. It decreased osteoclast marker enzyme TRAP, MMP-9, and Cathepsin K expression (P < 0.001). In vitro experiments demonstrated that miR-133b-3p-enriched exosomes promoted TGF-β1 expression and CD4 + CD25 + Foxp3+ Treg cell differentiation, while miR-133b-3p inhibitor exosomes had opposite effects.
Conclusion
Exosomal miR-133b-3p derived from BMSCs mitigates OP in rats, acting via the TGF-β1/Treg-mediated immune pathway, presenting a promising avenue for OP therapy.
Introduction
Osteoporosis (OP) is a skeletal disease characterized by decreased bone density, decreased amount of bone tissue, increased bone resorption, degradation of bone microstructure, and increased bone fragility [1]. The weakened bone strength predisposes individuals to osteoporotic fractures. These fractures not only amplify the likelihood of complications but also escalate the rates of disability and mortality, gravely impacting patients’ quality of life and exerting substantial economic strains on families and the society [2]. Researches have shown that factors like declining estrogen levels, aging, and excessive use of glucocorticoid drugs contribute to OP’s onset [3]. Although the pathogenesis of OP remains not entirely elucidated, the core pathological mechanisms involve an imbalance in bone homeostasis and a disruption of bone metabolism. This results in diminished osteoblast differentiation and heightened osteoclast activity, culminating in more bone resorption than formation [4]. Hence, the therapeutic crux for OP lies in fostering bone formation, curbing bone resorption, and restoring lost bone tissue.
Bone-marrow-derived mesenchymal stem cells (BMSCs) are mesoderm-derived stem cells with multidirectional differentiation potential. They can not only self-replicate and differentiate in multiple directions but also evolve into osteoblasts, adipocytes, cardiomyocytes, chondrocytes, among others [5,6]. Researches have shown that a compromised osteogenic differentiation ability or an enhanced adipogenic differentiation propensity in BMSCs has been linked to OP’s development [7].Thus, modulating the osteogenic differentiation of BMSCs has emerged as a focal area of OP research [8,9]. Exosomes, pivotal players in intercellular communication, antigen presentation, immunosuppression, and signaling, also influence tumor progression, metastasis, inflammation, and autoimmune diseases [10]. Some researchers discovered that exosomes derived from BMSCs bolster fracture healing and alleviate OP by stimulating angiogenesis, osteoblast proliferation, differentiation, and bone genesis [11]. This suggests that BMSCs-derived exosomes can be used as a therapeutic target for OP. Furthermore, miRNAs of from these exosomes can calibrate osteoblast or osteoclast differentiation and bone resorption activity. Manipulating these miRNAs-either by amplification or suppression-can enhance bone regeneration, facilitate fracture mending, and ameliorate OP [12,13]. However, the precise molecular dynamics of how BMSCs-derived exosomal miRNAs impact angiogenesis and osteogenesis are still not fully understood.
MicroRNAs (miRNAs) encased within exosomes can traverse extracellularly to modulate the biological functions of recipient cells [14]. By latching onto the 3ˊ-untranslated region (3ˊUTR) of target mRNAs, they can diminish the expression of certain genes, influencing vital cellular processes like development, proliferation, differentiation, and apoptosis [15]. Lately, the role of miRNAs in osteogenic differentiation has received extensive attention. Researchers have pointed out that miRNA-130a and miRNA-150-3p can bolster osteogenic differentiation of BMSCs and alleviate OP [16,17], positioning miRNAs as potential OP biomarkers and therapeutic targets. In addition, osteoblasts in mice can discharge exosomes enriched with osteogenic miRNAs [18]. Exosomes extracted from BMSCs of OP patients exhibited elevated miR-21 levels compared to their healthy counterparts. When these BMSCs were exposed to exosomes sourced from bone marrow stromal stem cells of OP patients, the exosomal miR-21 was observed to obstruct osteogenesis by targeting SMAD7 in vitro [19]. This suggests the regulatory influence of exosomal miRNAs on osteoblast functionality and differentiation. While miR-133b adjusts the osteogenic differentiation of adipose MSCs by targeting RUNX2 [20], in-depth inquiries into the potential role and mechanics of exosomal miR-133b-3p derived from BMSCs in relation to OP are warranted.
In our research, we extracted and characterized miRNAs from exosomes derived from BMSCs in osteoporotic rats. Animal experiments were conducted to fathom the modus operandi of miR-133b-3, a BMSCs-derived exosome, in counteracting OP. Our findings aim to offer fresh targets and a theoretical foundation for devising novel pharmaceuticals to combat OP. The detailed workflow of this study was shown in Fig. 1.
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Section snippets
Bioinformatics analysis of miRNA expression profiles
Microarray data (GSE64433) containing miRNAs expression profiles from pooled sera of osteopenia (n = 3) and osteoporosis (n = 3) patients were obtained from the Gene Expression Omnibus (GEO) database(https://www.ncbi.nlm.nih.gov/geo/). Raw data were processed using R software (version 4.2.0) with the limma package for quantile normalization. Differentially expressed miRNAs were identified using the criteria of fold change ≥1.3 and P < 0.05. Principal component analysis (PCA) was performed to
Bioinformatics analysis identifies miR-133b-3p as a key regulator in osteoporosis
To identify differentially expressed miRNAs associated with osteoporosis, we analyzed publicly available microarray data from the GEO database (GSE64433). This dataset contained miRNA expression profiles from pooled sera of osteopenia (n = 3) and osteoporosis (n = 3) patients. Quality control analysis confirmed the reliability of the dataset, with uniform distributions achieved after quantile normalization (Supplemental Fig. 1A-F).
Differential expression analysis revealed 27 significantly
Discussion
Bone health is underpinned by the dynamic equilibrium between osteoblasts and osteoclasts. Disruptions in this balance, often due to factors like aging, declining estrogen levels, or drug interventions, can predispose individuals to osteoporosis (OP) [21]. Present therapeutic modalities, including bisphosphonates, estrogens, romosozumab, and teriparatide, while effective, come with their own set of drawbacks, including adverse reactions and long-term complications [22]. Our research underscores
Conclusion
In conclusion, our study accentuates the therapeutic potential of BMSC-derived exosomes, particularly miR-133b-3p, in OP. These insights furnish a foundation for future investigations into the role of BMSC-derived exosomes in OP and other related disorders. However, this study was only conducted in an osteoporosis rat model and lacked relevant cellular level studies on the mechanism of BMSCs-derived exosomal miR-133b-3p in promoting osteogenic differentiation. In vitro and clinical studies are
CRediT authorship contribution statement
Yun Zhao: Writing – review & editing, Writing – original draft, Funding acquisition, Data curation. Xingyao Yang: Writing – review & editing, Writing – original draft, Data curation. Jialun Wang: Writing – review & editing, Conceptualization. Fangdong Chen: Writing – review & editing, Project administration. Guojia Shi: Writing – review & editing, Data curation. Jun Cheng: Writing – review & editing, Project administration, Conceptualization. Shuxing Xing: Writing – review & editing, Project
Ethics statement
All procedures were approved by the Ethics Committee of the Chengdu Fifth People’s Hospital. (Approval No.: 2023-013(Dept)-01).
Funding
This study was supported by the Key R&D Projects of Sichuan Provincial Science and Technology Programme (No. 2023YFS0235) and the Medical Scientific Research Project of Chengdu (No. 2023346).
Declaration of competing interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Acknowledgements
We thank all the participants for participating in our study.
References (39)
S. Song
Advances in pathogenesis and therapeutic strategies for osteoporosis
Pharmacol. Ther.
(2022)
J.H. Hwang
Yam-derived exosome-like nanovesicles stimulate osteoblast formation and prevent osteoporosis in mice
J. Control. Release
(2023)
F. Liu
S-sulfhydration of SIRT3 combats BMSC senescence and ameliorates osteoporosis via stabilizing heterochromatic and mitochondrial homeostasis
Pharmacol. Res.
(2023)
Y. Zhang
microRNA-935-modified bone marrow mesenchymal stem cells-derived exosomes enhance osteoblast proliferation and differentiation in osteoporotic rats
Life Sci.
(2021)
X. Yang
T cell-depleting nanoparticles ameliorate bone loss by reducing activated T cells and regulating the Treg/Th17 balance
Bioact Mater
(2021)
M.D. Walker et al.
Postmenopausal osteoporosis
N. Engl. J. Med.
(2023)
I. Foessl
Long-term and sequential treatment for osteoporosis
Nat. Rev. Endocrinol.
(2023)
I.R. Reid
A broader strategy for osteoporosis interventions
Nat. Rev. Endocrinol.
(2020)
M. Sikora
MiR-21-5p regulates the dynamic of mitochondria network and rejuvenates the senile phenotype of bone marrow stromal cells (BMSCs) isolated from osteoporotic SAM/P6 mice
Stem Cell Res Ther
(2023)
D. Li
The miR-4739/DLX3 axis modulates bone marrow-derived mesenchymal stem cell (BMSC) osteogenesis affecting osteoporosis progression
Front Endocrinol (Lausanne)
(2021)
H. Wang
LncRNA TCONS_00023297 regulates the balance of osteogenic and adipogenic differentiation in bone marrow mesenchymal stem cells and the coupling process of osteogenesis and angiogenesis
Front. Cell Dev. Biol.
(2021)
L. Hu
MiR-1224-5p modulates osteogenesis by coordinating osteoblast/osteoclast differentiation via the Rap1 signaling target ADCY2
Exp. Mol. Med.
(2022)
Y. You
WTAP-mediated m(6)A modification modulates bone marrow mesenchymal stem cells differentiation potential and osteoporosis
Cell Death Dis.
(2023)
R. Kalluri et al.
The biology, function, and biomedical applications of exosomes
Science (New York, N.Y.)
(2020)
J. Wang
Bone-targeted exosomes: strategies and applications
Adv. Healthc. Mater.
(2023)
F. Tan
Clinical applications of stem cell-derived exosomes
Signal Transduct. Target. Ther.
(2024)
R. Garcia-Martin
MicroRNA sequence codes for small extracellular vesicle release and cellular retention
Nature
(2022)
R.G. Avila-Bonilla et al.
Interactions of host miRNAs in the flavivirus 3´UTR genome: from bioinformatics predictions to practical approaches
Front. Cell. Infect. Microbiol.
(2022)
Z. Lin
Microrna-130a controls bone marrow mesenchymal stem cell differentiation towards the osteoblastic and adipogenic fate
Cell Prolif.
(2019)
