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

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.