Abstract
Background/Aim: This study aimed to explore the expression, function, and regulatory mechanism of exosomal miR-106b-5p in osteoporosis (OP), to identify a novel diagnostic biomarkers and therapeutic targets.
Materials and Methods: Plasma-derived exosomes were isolated in three independent case-control study samples (N=278, OP patients vs. healthy controls=139:139) to identify OP-associated miRNAs. Lentivirus-infected MC3T3-E1 and RAW264.7 cells were used to investigate the impact of upregulated miR-106b-5p on the proliferation, apoptosis, and differentiation of osteoblasts and osteoclasts, respectively. Target genes were predicted via bioinformatics.
Results: Exosomal miR-106b-5p abundance was screened to be consistently up-regulated with OP in the three sample sets. Overexpression of miR-106b-5p significantly affected proliferation, apoptosis, and cell cycle of MC3T3-E1 but not RAW264.7. RT-qPCR, ALP and TRAP staining showed that the upregulated miR-106b-5p significantly inhibits osteogenic differentiation and enhances bone resorption. Additionally, miR-106b-5p was confirmed to target and negatively regulate expression of SMAD5, BMP2, and MAPK1 genes.
Conclusion: Plasma exosomal miR-106b-5p promotes OP by targeting SMAD5/BMP2/MAPK1 to suppress bone formation and enhance resorption, representing a potential diagnostic biomarker and therapeutic target.
Introduction
Osteoporosis (OP) is a common metabolic bone disease characterized by decreased bone mass and degradation of the microstructure of bone tissue, resulting in increased bone fragility and susceptibility to fracture. Osteoporotic fractures in China are projected to grow to 6 million cases by 2050, costing $25.4 billion annually. Studies have shown that within a year after a hip fracture, 20% of patients may die from complications; about 50% will be disabled and have a significantly reduced quality of life (1-3).
Exosomes (Exos) are nanoparticles secreted by various cell types, ranging in size from 30-150 nm, which are released into the extracellular space and enter the circulatory system to exert local paracrine or distal endocrine effects (4-6). Exosomes serve as important carriers of intercellular and inter-tissue communication signals, including various components of cellular origin, such as DNA, RNA (mRNA, lncRNA, miRNA), lipids, and metabolites (7).
In human cells, there are over 2,300 different types of miRNAs, which are non-coding small RNA molecules approximately 18-25 nucleotides in length (8). A single miRNA can bind to the 3′ untranslated region (3′UTR) or 5′ untranslated region (5′UTR) of target gene mRNA, inhibiting the translation of the target mRNA and/or promoting its degradation, thereby regulating protein synthesis and downstream cellular pathways. It is estimated that up to 60% of protein-coding genes are post-transcriptionally regulated by miRNAs (9). Besides, miRNAs can also enter the nucleus and affect gene transcription. Furthermore, miRNAs are capable of regulating signal transduction, cell proliferation, and differentiation (10).
Previous studies demonstrated that exosomal miRNAs regulate function of recipient cells, can be used as disease biomarkers, and hold promise for reversing OP (11-14). For instance, mastocytosis-derived extracellular vesicles deliver miR-23a and miR-30a, which influence osteoblast function by targeting RUNX2 and SMAD1/5, preventing osteoblastogenesis and bone formation (15). Osteoclasts-derived exosomes target osteoblasts through EphrinA2, transferring miR-214 to inhibit osteogenic differentiation (16). Endothelial cell-derived exosomes have been shown to specifically target bone tissue, enhancing recovery from OP both in vitro and in vivo by delivering miR-155 (17). The use of exosomes as therapeutic agents is an emerging field, with studies like those by Hu et al., demonstrating the potential of human umbilical cord blood-derived exosomes (UCB-EVs) to increase bone mass and improve osteoporotic symptoms in mice by enhancing osteogenic effects through delivery of miR-3960 (18). These results collectively indicate that exosomes hold therapeutic promise for bone micro-injury repair and fracture prevention (19). In light of the rich contents of exosomes, the regulatory roles of miRNAs in bone metabolism-related cells such as osteoblasts and osteoclasts remain to be explored and elucidated.
To identify and characterize more plasma exosomal miRNAs associated with OP, the present study isolated plasma exosomes, screened for differentially expressed miRNAs and validated them in independent case-control study samples. Subsequently, the effects of a key miRNA, i.e., miR-106b-5p, on the proliferation, apoptosis, and differentiation of osteoblastic and osteoclastic cells was explored.
Materials and Methods
The workflow of the present study was presented in Figure 1 and detailed as follows.
Schematic illustration of the study design and workflow.
Patient selection. This study was based on the ongoing OP Prevention Project (OPP), a prospective community-based cohort in Suzhou, China, designed to identify OP risk biomarkers in elderly Chinese individuals. OP was defined by total hip bone mineral density (BMD) T-score <−2.5, measured by dual-energy X-ray absorptiometry (DEXA; Hologic Discovery, Marlborough, MA, USA). To minimize factors that might interfere with bone metabolism, strict exclusion criteria were implemented to exclude the following subjects: 1) Participants with chronic diseases or a history of conditions that could severely alter metabolic processes, such as diabetes mellitus, abnormal parathyroid functions (including hypothyroidism and hyperthyroidism), and thyroid disease, 2) patients with severe damage to vital organs such as the heart, lungs, liver, kidneys, and brain, 3) subjects with other bone-related disorders (e.g., Paget’s disease and rheumatoid arthritis), 4) subjects with a history of cancer (especially prostate, ovarian, and breast cancer), and 5) subjects with long-term use of medications known to affect bone metabolism (including corticosteroids and anticonvulsants). ALL the subjects were Chinese females. The project was approved by the Institutional Research Ethics Board at Soochow University (No: SUDA20200622H01), and all the participants had signed the informed consent before participating in the OPP project. Three case-control study samples were generated as follows (Table I).
Basic characteristics of the study samples.
Sample 1 (discovery): 9 OP patients and 9 age-matched controls. Every 3 random plasma samples from either the case group or the control group were mixed, and the resultant 6 mixed plasma samples were used for exosome isolation and the miRNA custom array experiment. Herein, a custom microarray (CT Bioscience, Jiangsu, PR China) with two plates (384-well format) covering a total of 768 miRNAs was used. RNA concentrations were quantified using a Qubit miRNA assay. Subsequently, in accordance with the manufacturer’s protocol, 100 ng of the RNA was reverse-transcribed into cDNA by means of a miRNA reverse transcription kit, with the utilization of stem-loop primers (20).
Sample 2 (replication): 30 OP patients and 30 age-matched controls.
Sample 3 (validation): 100 OP patients and 100 controls from an independent population.
Isolation and characterization of plasma exosomes. Exosomes were purified using the Qiagen exoRNeasy Serum/Plasma Midi Kit (QIAGEN, Hilden, Germany). Initially, plasma was filtered through a 0.22 μm filter to eliminate larger particles, followed by addition of an equal volume of buffer XBP to the plasma. The mixture was transferred to the exoEasy Spin Column, centrifuged at 500×g for 1 min, and the filtrate was discarded. Continued with Buffer XWP, centrifuged at 5,000×g for 5 min and discard the filtrate. Then the vesicles bound to the membrane were washed with Buffer XE, centrifuged at 500×g for 5 min and the eluents collected were plasma exosome samples. The morphological attributes of the exosomes were scrutinized via transmission electron microscopy (H-7650, Hitachi Ltd., Tokyo, Japan) as well as by the Zetasizer Nano ZS (Malvern, Worcestershire, UK). Additionally, Specific exosome markers were identified with fluorescently labeled antibodies (anti-CD63 and anti-CD81) by using flow cytometry (Beckman Coulter, Brea, CA, USA).
RNA extraction and RT-qPCR analysis. In Study Samples 2 and 3, total RNA from plasma exosomes was extracted using QIAzol (QIAGEN) reagent, whereas RNA from cultured cells (RAW264.7, MC3T3-E1) was isolated using TRIzol (ThermoFisher, Waltham, MA, USA) reagent, according to the respective manufacturers’ protocols. RNA concentration and purity were evaluated using an ultra-micro UV-visible spectrophotometer (Nanodrop 2000, ThermoFisher). RT-qPCR was performed using SYBR Green dye (Vazyme, Nanjing, PR China) to determine the expression levels of exosomal miRNAs, osteoblast marker genes (ALP, OPN, RUNX2, OCN), and osteoclast marker genes (CTSK, NFATC1, OSCAR, RANK, TRAP). Stem-loop primers were used for miRNA detection with U6 as the internal control, and mRNA levels were normalized to GAPDH. Relative expression was calculated using the 2−ΔΔCT method. The primer sequence was shown in Table II.
Primer sequences used for RT-qPCR.
Cell culture and transfection. Cells were purchased from the Institute of Cell Bank/Institutes for Biological Sciences (Shanghai, PR China, http://www.cellbank.org.cn). MC3T3-E1 cells were cultured in α-MEM containing 10% FBS and 1% penicillin/streptomycin; its differentiation medium was supplemented with 0.05 mM L-ascorbic acid, 10 mM β-glycerophosphate and 100 nM dexamethasone (all from Sigma-Aldrich, St. Louis, MO, USA) in 50 ml. RAW264.7 and HEK 293T cells were maintained in DMEM with 10% FBS and 1% penicillin/streptomycin, and RAW264.7 differentiation medium was added with 50 ng/ml RANKL. miR-106b-expressing lentiviral vectors were constructed by Shanghai Transheep, and co-transfected with packaging plasmids into HEK 293T cells. Viral particles were collected at 48 h post-transfection to infect MC3T3-E1 and RAW264.7 cells. The miR-106b overexpression sequence:
F 5′- GCTCTAGACCCACAGGCGTTACATAG-3′,
R 5′-GGAATTC TCAGCAGTAGGTTGGGTAAT-3′
Cell counting kit-8 assay (CCK-8). To evaluate cell proliferation, MC3T3-E1 and RAW264.7 cells, including untransfected cells, pTSB control cells, and cells with miR-106b overexpression, were seeded in 96-well plates at 5×103 cells per well (n=5). CCK-8 solution (10 μl) was added at 0, 12, 24, 36, 48, and 72 h, followed by 3 h incubation. Absorbance at 450 nm was measured using a microplate reader (Molecular Devices, San Jose, CA, USA).
Flow cytometry. Cell cycle and apoptosis were analyzed by flow cytometry in miR-106b overexpression, pTSB control cells, and untransfected MC3T3-E1 and RAW264.7 cells. Cell cycle was assessed using propidium iodide staining (Beyotime, Shanghai, PR China), and the percentages of G0/G1, S, and G2/M phases were determined. Apoptosis was detected using an Annexin V-FITC/PI kit (Takara, Shiga, Japan). Viable, early apoptotic, and late apoptotic/necrotic cells were distinguished and quantified.
Alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) staining. To test the effect of miR-106b-5p on osteoblast or osteoclast activity, cells were inoculated in 96-well plates at a density of 5×103 per well, three replicate wells were set up, and after seven days of differentiation culture, ALP staining for MC3T3-E1 was performed with NBT/BCIP solution (Beyotime), and Trap staining (Takara) was performed for RAW264.7. The cells were incubated for 30 min at room temperature before the dye was discarded. Then, the cells were washed three times with distilled water, and photographed using a fluorescence inverted microscope (Nikon, Tokyo, Japan).
Osteoclasts scratch assay and resorption pit formation assay. For the scratch assay, RAW264.7 cells were seeded in 24-well plates (1×105/well), scratched with a 10 μl tip, rinsed with PBS, and imaged at 0, 12, and 24 h to assess migration. For the resorption pit formation assay, cells were seeded (1×105/well) in Corning® Osteo Assay Surface plates (Corning Incorporated, Corning, NY, USA), cultured for 7 days, then removed with 10% sodium hypochlorite. After washing and drying, resorption pits were observed and counted under a microscope.
Identification of miR-106b-5p target genes. The target genes of miR-106b-5p were predicted through bioinformatics analyses with TargetScan (available at http://www.targetscan.org/) (21), miRDB (available at http://mirdb.org/) (22), and DIANA-microT-CDS (https://dianalab.ece.uth.gr/html/dianauniverse/index.php?r=microT_CDS) (23). The concatenated sets of target genes were subject to KEGG or GO pathway enrichment analysis.
mRNA sequencing. Library construction followed Illumina protocols. Total RNA (0.1 μg) with RIN ≥7 and 28S/18S ≥1.5 was used. Poly(A) mRNA was enriched with oligo(dT) beads, fragmented, and reverse-transcribed. Second-strand synthesis was performed at 16 °C. After end repair, dA-tailing, and adapter ligation, the library was PCR-amplified. Data were extracted with Agilent Feature Extraction (v10.7) and processed using GeneSpring GX (v12.0).
Statistical analysis. Student’s t-test was used for mean comparisons of the two groups, and one-way ANOVA was used for multiple comparisons. Receiver operating curves (ROC) were constructed and the area under the curve (AUC) with a 95% confidence interval was calculated to evaluate the diagnostic accuracy of miRNAs. Spearman correlation analysis was used to estimate the relationship between miR-106b-5p abundance and clinical parameters. The software R4.2.3 was used to perform the statistical analyses. p<0.05 was considered statistically significant.
Results
Plasma exosomal miR-106b-5p is up-regulated OP. Exosomes were isolated from plasma, and electron microscopy and nanoparticle analyses showed that the exosomes were circular and about 100 nm in diameter (Figure 2). Next, RT-qPCR-based microarrays were used to profile 768 exosomal miRNAs in plasma from 9 OP patients and 9 age-matched controls; samples within each group were randomly pooled into 3 replicates. Among 256 detectable miRNAs (CT<35), 11 candidate miRNAs were selected at p<0.1, including miR-106b-5p (log2FC=0.53, p=0.049; Figure 3A). The upregulation of exosomal miR-106b-5p in OP was further validated by RT-qPCR in two independent cohorts: Sample 2 (log2FC=0.8, p=0.036; Figure 3B) and Sample 3 (log2FC=0.99, p<0.001; Figure 3C).
Characteristics of the isolated exosome samples. (A) Representative TEM image of the isolated plasma-derived exosome sample. (B) NTA result showing the size distribution of the isolated plasma-derived exosome sample.
Plasma exosomal miR-106b-5p is associated with OP. (A) The fold change (FC) values of exosomal miRNAs with significant differences in case vs. control in the study sample 1 (N=18, case vs. control=9:9). Threshold of significance: p<0.1; miR-106-5p: log2FC=0.53, p=0.049. (B) Up-regulation of plasma exosomal miR-106b-5p in OP in Sample 2 as determined by RT-qPCR (p=0.036; N=60, case vs. control=30:30). (C) Up-regulation of plasma exosomal miR-106b-5p in OP in Sample 3 as determined by RT-qPCR (p<0.001; N=200, case vs. control=100:100). (D) ROC curve showing the diagnostic performance of plasma exosomal miR-106b-5p after correcting for age and BMI (AUC=0.71). (E) Correlation of plasma exosomal miR-181a-5p abundance with hip BMD T-score (r=−0.228, p=0.0012). (F) Correlation of plasma exosomal miR-181a-5p abundance with clinical parameters. *p<0.05, **p<0.01, ***p<0.001. OP: Osteoporosis; ROC: receiver operating characteristic.
To evaluate the diagnostic potential of exosomal miR-106b-5p for OP, we performed ROC curve analysis using combined data from Sample 2 and Sample 3. After adjusting for age and BMI, miR-106b-5p effectively distinguished OP patients from controls (case vs. control:130:130, AUC=0.71) (Figure 3D). In the combined cohort (N=260), miR-106b-5p was inversely correlated with hip BMD T-score (r=−0.23, p=0.001, Figure 3E) and remained independently associated with OP in logistic regression (p=0.007). It was positively correlated with PLR and negatively correlated with lymphocyte count (r=−0.18, p=0.005) and hematocrit (r=−0.13, p=0.046), suggesting a role in immune and inflammatory processes (Figure 3F).
Up-regulation miR-106b-5p inhibits osteoblast differentiation. MC3T3-E1 cells were infected with miR-106b-overexpressing or pTSB negative control lentivirus, resulting in a 6.77-fold upregulation of miR-106b-5p (Figure 4A). Proliferation of MC3T3-106 cells was comparable to control groups within 24 h but significantly decreased thereafter (Figure 5A), while apoptosis showed no differences (Figure 5B). miR-106b overexpression increased G0/G1 phase and decreased S phase cell proportion (p=0.01), inhibiting G0/G1-to-S phase transition (Figure 5C). ALP staining (Figure 5D) and RT-qPCR showed reduced osteogenic differentiation in MC3T3-106 cells (ALP: FC=0.52; OCN: FC=0.04; OPN: FC=0.31; Runx2: FC=0.60) (Figure 5E), indicating miR-106b-5p inhibits osteoblast proliferation and osteogenic differentiation.
Lentivirus overexpressing miR-106 infects MC3T3-E1 and RAW264.7 cells. (A) Over-expression of miR-106b in MC3T3-E1 cells after lentiviral transfection (FC=6.77), as determined by RT-qPCR. (B) Over-expression of miR-106b in RAW264.7 cells after lentiviral transfection (FC=10.56), as determined by RT-qPCR.
Overexpression of miR-106b-5p inhibits osteoblastic bone formation. (A) MC3T3-E1 cells exhibited a declined survival rate upon miR-106b transfection. (B) The impact of lentiviral transfection on apoptosis in MC3T3-E1 cells. (C) The impact of miR-106b overexpression on the cell cycle of MC3T3-E1 cells (G0/G1 phase: p=0.01; S phase: p=0.03). (D) Inhibited osteoblast activity in MC3T3-E1 cells after miR-106b transfection as determined by ALP staining. (E) Inhibited osteogenic gene expression in MC3T3-E1 cells after miR-106b transfection as determined by RT-qPCR.
miR-106b-5p enhances osteoclastic bone resorption. After lentiviral infection, the abundance of miR-106b-5p in the RAW-106 group increased significantly (FC=10.56) (Figure 4B). No significant differences in cell proliferation, apoptosis, or cell cycle were observed among the three groups (Figure 6A-C). However, the scratch assay revealed a slightly faster closure rate in the miR-106b overexpressing cells compared to the NC group, suggesting that miR-106b overexpression enhances the migratory ability of RAW264.7 cells (Figure 6D). Corning Osteo Assay revealed more bone resorption pits in the RAW-106 group than in control and RAW-NC cells (Figure 6E), suggesting miR-106b overexpression promotes osteoclastic bone resorption. TRAP staining showed RAW-106 and RAW-NC cells had greater differentiation than RAW264.7 cells (Figure 6F). RT-qPCR demonstrated significantly elevated expression of CTSK (FC=1.56, p<0.05), OSCAR (FC=2.49, p<0.05), and TRAP (FC=1.69, p<0.05) in RAW-106 cells compared to RAW-NC cells after differentiation (Figure 6G).
miR-106b-5p enhances osteoclastic bone resorption. (A) No variation occurred in the survival of RAW264.7 cells subsequent to miR-106b transfection. (B) No significant impact of lentiviral transfection on RAW264.7 cells apoptosis. (C) RAW264.7 cells cycle unaffected by miR-106b overexpression. (D) The miR-106b overexpression promotes migration of RAW264.7 cells. (E) The miR-106b overexpression enhances bone resorption activity of osteoclasts. (F) Osteoclast differentiation of RAW264.7 cells transfected with miR-106b vs. NC, as determined by TRAP staining. (G) Increased expression of bone resorption-related genes in RAW264.7 cells transfected with miR-106b vs. NC, as determined by RT-qPCR. NC: pTSB control cells.
The miR-106b-5p directly targets the SMAD5/BMP2/MAPK1 genes. KEGG enrichment analysis of miR-106b-5p target genes showed enrichment in OP-related pathways (MAPK, TGF-β, Wnt, TNF; Figure 7A). There were 393 overlapping target genes of miR-106b-5p in the three databases (Figure 7B). After further literature review, we selected RANKL, MMP2, SMAD5, BMP2, MAPK1 as candidate target genes of miR-106b-5p for validation. RT-qPCR showed SMAD5 (FC=0.47), BMP2 (FC=0.22), and MAPK1 (FC=0.01) were downregulated in MC3T3-106 cells (Figure 7C), suggesting miR-106b-5p inhibits osteoblast differentiation by suppressing these genes.
miR-106b-5p gene screening and validation. (A) KEGG pathways significantly enriched by target genes of miR-106b-5p. (B) Venn diagram illustrating the putative candidate target genes of miR-106b-5p predicted by miRDB, TargetScan, and DIANA-microT-CDS. (C) mRNA expression level of miR-106b-5p target genes. (D) Volcano plot showing differential mRNA expression. (E) GO categories significantly enriched by differentially expressed genes.
Transcriptome sequencing of MC3T3-106 and MC3T3-NC cells identified 293 upregulated and 380 downregulated DEGs (|log2 (FC)| ≥1, p<0.05; Figure 7D). GO enrichment showed DEGs were enriched in bone metabolism-related processes (e.g., bone mineralization, positive regulation of osteoblast differentiation; Figure 7E). Among these experimentally detected DEGs, 35 genes coincided with the bioinformatically predicted target genes, including SMAD5 and MAPK1, which were verified by RT-qPCR analysis (SMAD5: FC=0.40, p=2.51E-24; MAPK1: FC=0.44, p=2.21E-02).
Discussion
The equilibrium of regulatory activity between bone-forming osteoblasts and bone-resorbing osteoclasts is essential for bone repair (24). We screened differential plasma exosomal miRNAs in three independent sample sets and evaluated their diagnostic potential; ROC analysis showed plasma exosomal miR-106b-5p effectively distinguished OP patients from healthy individuals. miR-106b-5p inhibited osteogenic differentiation, as its upregulation reduced mRNA expression of ALP, OCN, OPN, RUNX2 and ALP activity in vitro. It also enhanced osteoclast-mediated bone resorption and upregulated CTSK, OSCAR, TRAP mRNA. Collectively, these findings suggest that reducing miR-106b-5p may promote osteogenesis. This aligns with recent findings that preserving osteoblast function, whether by targeting miRNAs or cholesterol metabolites such as 25-hydroxycholesterol, represents a key therapeutic strategy for OP (25).
Reports indicate that exosomes from mammalian cells contain more than 1,600 types of mRNA and 700 varieties of miRNA, with miRNAs identified as crucial regulators in the intricate control of bone coupling (19, 26). Zhang et al. found that miR-22-3p in bone marrow mesenchymal stem cells-derived Exos promotes osteogenic differentiation via FTO inhibition (27). Enhanced miR-135b abundance in mesenchymal stem cells-derived Exos down-regulates Sp1 expression in chondrocytes to promote chondrocyte proliferation (28). Exosomes may vary according to the phenotype and function of the source cells, and even the exosomes secreted by the same cells in different states have different compositions and functions (29). Some studies have found that the proportion of miRNAs in exosomes is even higher than that in cells of origin, so it is believed that miRNAs are not randomly integrated into exosomes (30). Currently, specific changes in exosomal miRNA can be detected in certain diseases, suggesting that exosomal miRNA can be used as a biomarker for disease diagnosis (14, 31-33). Our study identified that higher abundance of miR-106b-5p in plasma-Exos may promote OP, and AUC=0.7, with good predictive performance and potential as a biomarker for OP diagnosis. miR-106b-5p is a member of the miR-106b-25 cluster, located within the 13th intron of the MCM7 gene on the long arm of human chromosome 7, region 22. Previous studies have suggested that miR-106b-5p serves as a potential marker for predicting early metastasis after nephrectomy in patients with renal cell carcinoma (9). Additionally, it can influence the invasion of breast cancer cells by regulating the expression of its target gene MMP2 (34). In the context of OP, miR-106b-5p has a negative regulatory effect on the osteogenic differentiation of mesenchymal stem cells from the placenta (PMSCs) (35). It is suggested that miR-106b may influence bone resorption, as inhibiting miR-106b in mice can reduce the levels of C-terminal telopeptide of type I collagen (CTX-1) in the serum. Type I collagen accounts for over 90% of the organic bone matrix. During bone turnover, type I collagen is degraded, and short peptide fragments are released into the bloodstream, serving as an indicator of bone resorption. CTX-1 has long been used in the clinic for the detection and assessment of bone absorption in postmenopausal women with OP, as well as for predicting the effectiveness of anti-resorptive therapies (36). However, overall, the role of miR-106b-5p in OP still lacks systematic research. The sequences of miR-106b- 5p are identical in mice and humans. Therefore, current results hold significant potential in the development of therapies for OP.
To elucidate the molecular mechanisms of miR-106b in regulating osteoblast differentiation and bone formation, we have assessed the potential of BMP2, MAPK1, MMP2, and SMAD5 to serve as target genes for miR-106b-5p. Corresponding to the findings of this study, it has been reported that the expression of SMAD1 and SMAD5 genes in the femurs of osteoporotic mice is lower than in normal mice. Inhibitors of miR-106b can upregulate the expression of these two genes, suggesting that miR-106b-5p suppresses osteogenic differentiation by targeting SMAD5 and SMAD1 (37). Additionally, research has demonstrated that miR-106b influences osteogenic differentiation and bone formation of mesenchymal stem cells from bone marrow by targeting BMP2 (35). KEGG enrichment analysis of miRNA target genes, as well as transcriptome profiling of MC3T3-E1 cells, consistently highlight the MAPK signaling pathway. Numerous studies have confirmed that activating the MAPK signaling pathway can promote the differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts while inhibiting their differentiation into adipocytes, which holds potential positive implications for the treatment of OP (38). Our research has determined that elevated levels of miR-106b-5p lead to a decrease in the expression of MAPK1, a gene associated with bone metabolism. This suggests that inhibiting miR-106b may be a novel therapeutic strategy for the treatment of OP.
Conclusion
In summary, this study uncovers the role of exosomal miR-106b-5p in the pathology of OP, marking the initial discovery of its regulatory function between osteoblasts and osteoclasts. It was found that miR-106b significantly reduces the expression levels of multiple genes related to bone metabolism, such as SMAD5/BMP2/MAPK1, consequently contributing to reduced bone density and the onset of OP. These findings indicate that miR-106b-5p could potentially serve as a biomarker for the diagnosis and treatment of OP, offering new therapeutic options for exosomal targeting in the future.
Footnotes
Authors’ Contributions
All Authors contributed to the study conception and design. SFL, FYD, and YHL conceived the study; YHL, MHX, and LFW performed the experiments and analyzed the data; FYD, SFL, and MHX wrote the manuscript, and all Authors approved the final version of the manuscript.
Conflicts of Interest
Ming-Hui Xia, Yi-Hua Lu, Long-Fei Wu, Fei-Yan Deng, Shu-Feng Lei declare that they have no conflicts of interest.
Funding
The study was supported by National Natural Science Foundation of China (82373587, 82173529, 82173598, and 82103922) and a Project of the Priority Academic Program Development of Jiangsu Higher Education Institutions. The authors would like to acknowledge the financial support for this study from the National Key R&D Program of China, MOST (2023YFC2509900).
Artificial Intelligence (AI) Disclosure
No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.
- Received January 15, 2026.
- Revision received February 24, 2026.
- Accepted March 6, 2026.
- Copyright © 2026 The Author(s). Published by the International Institute of Anticancer Research.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
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