BMSC-derived exosomal miR-27a-3p and miR-196b-5p regulate bone remodeling in ovariectomized rats

Abstract

Background: In the bone marrow microenvironment of postmenopausal osteoporosis (PMOP), bone marrow mesenchymal stem cell (BMSC)-derived exosomal miRNAs play an important role in bone formation and bone resorption, although the pathogenesis has yet to be clarified.

Methods: BMSC-derived exosomes from ovariectomized rats (OVX-Exo) and sham-operated rats (Sham-Exo) were co-cultured with bone marrow-derived macrophages to study their effects on osteoclast differentiation. Next-generation sequencing was utilized to identify the differentially expressed miRNAs (DE-miRNAs) between OVX-Exo and Sham-Exo, while target genes were analyzed using bioinformatics. The regulatory effects of miR-27a-3p and miR-196b-5p on osteogenic differentiation of BMSCs and osteoclast differentiation were verified by gain-of-function and loss-of-function analyses.

Results: Osteoclast differentiation was significantly enhanced in the OVX-Exo treatment group compared to the Sham-Exo group. Twenty DE-miRNAs were identified between OVX-Exo and Sham-Exo, among which miR-27a-3p and miR-196b-5p promoted the expressions of osteogenic differentiation markers in BMSCs. In contrast, knockdown of miR-27a-3p and miR-196b-5p increased the expressions of osteoclastic markers in osteoclast. These 20 DE-miRNAs were found to target 11435 mRNAs. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that these target genes were involved in several biological processes and osteoporosis-related signaling pathways.

Conclusion: BMSC-derived exosomal miR-27a-3p and miR-196b-5p may play a positive regulatory role in bone remodeling.

Keywords: Bone marrow mesenchymal stem cell; Exosomes; MicroRNAs; Next-generation sequencing; Osteoclast; Osteogenic differentiation; Postmenopausal osteoporosis.

Figure 1. Evaluation of the osteoporotic rat model and identification of BMSCs.

(A) BMD, Tb. N, BV/TV, BS/TV, Tb. Sp and Tb. Pf of the fourth lumbar vertebrae from Sham and OVX rats. (B) Micro-CT three-dimensional view of the fourth lumbar vertebrae from Sham and OVX rats. (C) Evaluation of osteogenic differentiation of BMSCs by alizarin red staining (scale bars = 300 µm). (D) Evaluation of adipogenic differentiation of BMSCs by Oil Red O staining (scale bars = 200 µm). (E) The expressions of CD29, CD90, CD45 and CD11b in BMSCs are analyzed by flow cytometry. Data are presented as the mean ± SD, n = 3 rats/group. * P < 0.05, ns: not significant.

Figure 2. Characterization of BMSC-derived exosomes.

(A) Transmission electron microscopy image showing the shape and size of OVX-Exo and Sham-Exo (scale bars = 200 nm). (B) Western blot indicats the expression of CD81, Hsp70, TSG101 and Calnexin in cellular and exosomal lysates of BMSCs. (C and D) Particle size distribution of OVX-Exo and Sham-Exo using nanoparticle tracking analysis.

Figure 3. OVX-Exo and Sham-Exo are internalized by BMMs, affecting osteoclast differentiation.

(A and B) TRAP staining of the control groups and M-CSF+RANKL induction group (scale bars = 200 µm). (C) The mRNA levels of ACP5, CTSK and NFATc1 in the M-CSF+RANKL induction group compared to the control group determined by qRT-PCR. (D) The mRNA levels of ACP5, CTSK and NFATc1 in osteoclasts cocultured with OVX-Exo compared to Sham-Exo determined by qRT-PCR. (E) Confocal microscopy images show that OVX-Exo and Sham-Exo are internalized by BMMs (scale bars = 30 µm). Exosomes are labeled with PKH26 (red); nuclei are labeled with DAPI (blue); and cytoskeletons are labeled with phalloidin (green). β-actin is used for normalization of mRNA. Data are presented as the mean ± SD, and all experiments were repeated three times. ** p < 0.01, *** P < 0.001.

Figure 4. DE-miRNAs in OVX-Exo and Sham-Exo.

(A) Hierarchical clustering of the DE-miRNAs. Red pixels represent high expression, and blue pixels represent low expression. The original expression values of the miRNAs are normalized using the Z-score. The absolute strength of the signal ranges from −1.5 to +1.5, and the corresponding color ranges from blue to red. (B) Volcano map showing the distribution of DE-miRNAs, with rno-miR-27a-3p and rno-miR-196b-5p specially annotated.

Figure 5. Bioinformatics analysis of target genes of DE-miRNAs.

(A) The number of target genes predicted by DE-miRNAs. (B) The number of miRNAs targeting the target genes. (C) GO annotation of target genes. The 10 most enriched GO terms are listed with respect to biological process, cellular component, and molecular function based on p-values. (D) The 20 most common KEGG pathways of target mRNAs of DE-miRNAs.

Figure 6. MiR-27a-3p and miR-196b-5p promote osteogenic differentiation of BMSCs.

(A) Relative expression of miR-27a-3p in BMSCs from OVX rats compared with Sham rats. (B) Relative expressions of miR-27a-3p in BMSCs on day 5, 7 and 9 after osteogenesis induction compared with preinduction. (C) Relative expressions of miR-27a-3p in BMSCs transfected with miR-27a-3p mimic/inhibitor. (D–F) The protein and mRNA levels of ALP, OCN, OSX and RUNX2 in BMSCs transfected with miR-27a-3p mimic/inhibitor. (G) Relative expression of miR-196b-5p in BMSCs from OVX rats compared with Sham rats. (H) Relative expressions of miR-196b-5p in BMSCs on day 5, 7 and 9 after osteogenesis induction compared with preinduction. (I) Relative expressions of miR-196b-5p in BMSCs transfected with miR-196b-5p mimic/inhibitor. (J–L) The protein and mRNA levels of ALP, OCN, OSX and RUNX2 in BMSCs transfected with miR-196b-5p mimic/inhibitor. The expression levels of miRNA and mRNA were determined by qRT-PCR, and protein expressions were detected by western blot. U6 is used for the normalization of miRNA and β-actin is used for normalization of mRNA. β-actin served as the loading control in the western blot analysis. Data are presented as the mean ± SD, and all experiments were repeated three times. * P < 0.05, ** p < 0.01, *** P < 0.001, ns: not significant.

Figure 7. Knockdown of miR-27a-3p and miR-196b-5p promote osteoclast differentiation.

(A and B) Relative expression of miR-27a-3p and miR-196b-5p in BMSC-derived exosomes from OVX rats compared with Sham rats. (C and D) Relative expression of miR-27a-3p and miR-196b-5p in osteoclasts at day 4 after osteoclast differentiation induction compared with preinduction. (E) Relative expression of miR-27a-3p in osteoclasts transfected with miR-27a-3p inhibitor compared to inhibitor-NC. (F) The mRNA levels of ACP5, CTSK and NFATc1 in osteoclasts transfected with miR-27a-3p inhibitor compared to inhibitor-NC. (G) Relative expression of miR-196b-5p in osteoclasts transfected with miR-27a-3p inhibitor compared to inhibitor-NC. (F) The mRNA levels of ACP5, CTSK and NFATc1 in osteoclasts transfected with miR-196b-5p inhibitor compared to inhibitor-NC. Expression levels were determined by qRT-PCR. Cel-miR-39-3p and U6 are used for exosomal and cellular miRNA normalization, respectively. β-actin is used for mRNA normalization. Data are presented as the mean ± SD, and all experiments were repeated three times. * P < 0.05, ** p < 0.01, *** P < 0.001.