Exosomal miR-133b-3p modulates TGF-β1/Treg immunomodulation to ameliorate osteoporosis
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.