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

Experimental animals

A total of 18 Sprague-Dawley rats (female, 12 weeks old, weighing 220 ± 10 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All experimental animal procedures were approved by the Animal Ethics Committee of Shenzhen Hospital of Southern Medical University, China (approval No. 2021-0057). The rats were maintained on a 12-h light/dark cycle at 22 ° C with 50–55% humidity, and got free access to food and water. After one week of acclimatization, the rats were equally and randomly assigned to the OVX and Sham groups (9 rats per group). The rats were anesthetized by an intraperitoneal injection of 2% 2,2,2-tribromoethanol (Macklin, Shanghai, China). Then, bilateral ovaries were removed from the OVX rats, while peripheral ovarian fat was removed from the Sham rats, as described previously (Wu et al., 2021). After completion of operation the resuscitated rats were kept in the same conditions as before operation. To evaluate the rat PMOP model, rats were euthanized by cervical decoupling 12 weeks after the operation, and the fourth lumbar vertebrae (L4) were scanned using micro-CT (SkyScan 1176; Bruker, Kontich, Belgium) and analyzed for bone microstructure. The parameters were as follows: voxel size 10.139475 µm, medium resolution, 85 kV, 200 µA, one mm Al filter and integration time 384 ms. The data were analyzed using the manufacturer ’s evaluation software, including SkyScan NRecon (version 1.7.4.2), CTvox (version 3.3.0) and CTAn (version 1.18.8.0).

Culturing and identification of BMSCs

BMSCs were isolated from the femur and tibia of OVX and Sham rats and cultured in growth medium, as described previously (Xu et al., 2018a; Xu et al., 2018b). Briefly, the bilateral femurs and tibia were harvested under sterile conditions, and the bone marrow was collected. BMSCs were cultured in 10-mm Petri dish and replaced with the growth medium every other day. The growth medium included α-minimum essential medium (α-MEM), 10% fetal bovine serum (FBS), and 1% penicillin and streptomycin solution (Gibco, Carlsbad, CA, USA). BMSCs were passaged when the cell density reached 80–90% confluency.

Flow cytometric characterization was performed as described previously (Wu et al., 2021). Briefly, the passage 3 BMSCs were digested with 0.25% trypsin and suspended to a concentration of 1 × 10⁶ cells/mL with cell staining buffer (BioLegend, San Diego, CA, USA). Next, the cells were incubated with antibodies against CD29-phycoerythrin (PE; #102207), CD90-PE (#202207), CD11b-PE (#201807), or CD90-PE (#202523, BioLegend, San Diego, CA, USA) in the dark at 4 °C for 45 min. A tube of cells suspended in PBS was set as the control group. Subsequently, the cells were collected by centrifugation (4 °C, 150 × g, 5 min), and the cells were analyzed by flow cytometry on a Sony SA3800 apparatus (Sony Biotechnology, Tokyo, Japan).

For osteogenic differentiation of BMSCs, the cells were cultured in osteogenic induction medium (Cyagen Biosciences, Guangzhou, China) for 14 days and stained with alizarin red. For adipogenic differentiation, the cells were stained with Oil Red O after 3 weeks of culturing in adipogenic induction medium (Cyagen Biosciences).

Osteoclast culture and differentiation induction

For the isolation of bone marrow-derived macrophages (BMMs), bone marrow cells (BMCs) were lysed with erythrocyte lysate buffer (Leagene, Beijing, China) and centrifuged at 1200 rpm/min for 5 min. After removal of the supernatant, BMCs were cultured overnight in complete medium (α-MEM + 10% FBS), and then the unadhered cells were collected and cultured for 2 days in complete medium supplemented with 30 ng/mL macrophage colony stimulating factor (M-CSF) (PeproTech, Hamburg, Germany). BMMs were then inoculated into 96-well culture plates (8000 cells/well) and cultured in complete medium containing 30 ng/mL M-CSF and 50 ng/mL receptor activator ligand of NF-kB (RANKL) (PeproTech) for 4 days (the medium was changed every 2 days). Tartrate-resistant acid phosphatase (TRAP) activity was detected with a TRAP Stain Kit (Sigma-Aldrich, St. Louis, MI, USA) according to the manufacturer’s protocol, and the mRNA of osteoclastic markers were detected by real-time quantitative reverse transcription-PCR (qRT-PCR).

Isolation and characterization of BMSC-derived exosomes

When passage 3 BMSCs reached a confluency of 80–90%, the culture medium without exosomes was replaced, and the supernatant was collected after 2 days. The exosomes in the supernatant of BMSCs were extracted by differential ultracentrifugation (Rotor: SW 32 Ti, Beckman Coulter, Brea, CA, USA). Briefly, the supernatant was collected by centrifugation at 300, 2000, and 10,000 × g for 10, 10, and 30 min to remove the cells and cell debris. Next, protein contaminants were removed by ultracentrifugation of the supernatant at 110,000 × g for 70 min twice, and the precipitate was resuspended in PBS buffer. Subsequently, exosomes in the precipitate were resuspended in 100 µL PBS and stored at −80 ° C (Théry et al., 2006; Chen et al., 2019).

The approximate concentration and diameter of the exosomes were measured by nanoparticle tracking analysis (NTA) using a ZetaView Particle Metrix (Particle Metrix, Meerbusch, Germany). The shape and size of the exosomes were observed by transmission electron microscopy (TEM) (FEI Tecnai G2 Spirit BioTwin; FEI, Hillsboro, OR, USA). The exosome positive markers (CD81, Hsp70, and TSG101) and purity control (Calnexin) were detected by Western blotting.

Isolation of exosomal total RNA and miRNA sequencing

BMSC-derived exosomal total RNA was extracted using TRIzol (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The integrity of total RNA was analyzed by an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and RNA integrity number (RIN) >7 was used for NGS. The concentration of total RNA was detected by NanoDrop™ 2000 (Thermo Fisher Scientific) (Pang et al., 2020). As described previously, miRNA sequencing was performed on the Illumina HiSeq 2500 platform from LC Sciences (Hangzhou, China) (Pang et al., 2019). The raw reads of each sample were internally programmed by ACGT101-miR, and clean reads were obtained by removing the adapter dimers, junk, low complexity, common RNA families (rRNA, tRNA, snRNA, and snoRNA), and repetition. Then, unique sequences of 18–26 nucleotides were mapped to the precursors of Rattus norvegicus in miRBase 22.0 to identify the known and novel miRNAs. L/R ±  n indicated that the detected miRNA sequence was n bases more/less than the known miRNAs on the left/right side. The read counts corresponding to tags per million counts (TPM) ratio was used to normalize the expression levels of miRNAs. DE-miRNAs were identified by fold-change ≥2 or fold-change ≤0.5 and P ≤ 0.05.

Target gene prediction, GO and pathway enrichment analysis

To study the possible functions of DE-miRNAs, miRanda and TargetScan algorithms were used to predict their target genes that were subsequently removed with a context score percentile <50 in the TargetScan algorithm and a max energy value > −10 in the miRanda algorithm. Finally, those found at the intersection of the two algorithms were considered the final target genes of DE-miRNAs. The Gene Ontology (GO) project (http://www.geneontology.org) was used to analyze the gene function of the target genes of DE-miRNAs. We identified the significant pathways associated with enriched DE-miRNAs according to KEGG analysis (http://www.genome.jp/kegg). Fisher’s exact test and χ² test were used to classify the GO categories and KEGG pathways, and the false discovery rate (FDR) was calculated to correct the P-value. The thresholds of P < 0.05 and FDR < 0.05 were used to select the significant GO categories and KEGG pathways.

Exosome uptake assay

OVX-Exo and Sham-Exo were incubated with PKH26 (Sigma) for 5 min, and then the fluorescently labeled exosomes were centrifuged at 10,000 × g for 30 min and the supernatant was collected to remove the unbound dye. Subsequently, the exosomes were collected by 110,000 × g ultracentrifugation for 70 min. BMMs were cocultured with PKH26-labeled exosomes for 24 h and fixed with 4% paraformaldehyde. In the blank control group, exosomes were replaced with an equal volume of PBS. Cell cytoskeleton and nucleus were labeled with phalloidine and DAPI (Solarbio, Beijing, China), respectively, and observed under an Olympus FV1200 confocal microscope (Olympus, Tokyo, Japan).

Cell transfection

When the confluence of BMSCs and BMMs reached 50–60%, cells were transfected with mimic-NC, miR-27a-3p mimic, miR-196b-5p mimic, inhibitor-NC, miR-27a-3p inhibitor or miR-196b-5p inhibitor (RiboBio, Guangzhou, China) using Lipofectamine™ RNAiMAX Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The final concentration of miRNA mimic and mimic-NC were 100 nM, and miRNA inhibitor and inhibitor-NC were 200 nM. After 12 h of transfection, the transfection reagent was replaced with osteogenic induction medium or osteoclast induction medium. After 48 h of induction, mRNAs and proteins of osteogenic or osteoclastic markers were detected by qRT-PCR and Western blot, respectively.

qRT-PCR

Total RNA of cells and exosomes was extracted by TRIzol (Thermo Fisher Scientific). When the exosomes were fully lysed, 1 pmol of cel-miR-39-3p standard RNA was added. For miRNA detection, total RNA was reverse transcribed and amplified using the miDETECT A TrackTM miRNA qRT-PCR Starter Kit (RiboBio). For mRNA detection, total RNA was reverse transcribed with a TransScript All-in-One First Strand cDNA Synthesis SuperMix kit (TransGen Biotech, Beijing, China). PerfectStart Green qPCR SuperMix kit (TransGen) was used for quantitative real-time PCR (qPCR) on a LightCycler 96 instrument (Roche, Basel, Switzerland). The relative quantification was carried out by the 2^−ΔΔCT method. Cel-miR-39-3p was used as an external control for the detection of exosomal miRNAs, and U6 and β-actin were used as internal controls for cellular miRNA and mRNA detection, respectively. Cel-miR-39-3p standard RNA (Cat. miRB0000010), cel-miR-39-3p primer (Cat. miRA0000010) and U6 primer (Cat. miRAN0002) were purchased from RiboBio. Primer sequences for qRT-PCR are listed in Table S1.

Western blot

Total proteins were extracted from the cells and exosomes using the Whole Cell Lysis Assay (KeyGen Biotech, Nanjing, China), and the protein concentration was determined by the FD™ BCA Protein Quantitative Kit (Fdbio Science, Hangzhou, China). The lysates were separated by 10% SDS-PAGE (Fdbio Science), and the proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). Then, the blotting membrane was blocked with 5% milk in tris buffered saline with Tween 20 (TBST; Fdbio Science) at room temperature for 1 h, followed by probing with rabbit anti-CD81 (ab109201; Abcam, Cambridge, UK; 1:1,000), anti-TSG101 (ab125011; Abcam; 1:1,000), anti-Hsp70 (ab181606; Abcam; 1:1,000), anti-Calnexin (ab22595, Abcam; 1:1,000), anti-ALP (DF6225; Affinity Biosciences, Jiangsu, China; 1:500), anti-OCN (sc-390877; Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:100), anti-OSX (ab209484; Abcam; 1:1,000), anti-RUNX2 (ab236639; Abcam; 1:1,000) or anti-beta-actin (KC-5A08; Kangcheng Biotech, Shanghai, China; 1:10,000) antibodies, at 4 ° C overnight. Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-coupled goat anti-rabbit IgG H&L (ab205718; Abcam; 1:10,000) or goat anti-mouse IgG H&L (HRP) (ab205719; Abcam; 1:10,000). Signals were detected with the Immobilon Western Chemiluminescent HRP substrate (Millipore) and recorded on an X-ray film (Estman Kodak Company, XBT-1).

Statistical analysis

Statistical analysis was carried out using SPSS 25.0 software (IBM Corp., Armonk, NY, USA.), and P < 0.05 indicated statistical significance. Measurement data were expressed as the mean ± standard deviation (SD). Normally distributed data and homogeneity of variance between the two groups were compared using unpaired t-tests.

Characterization and miRNA expression of BMSC-derived exosomes

TEM analysis showed that OVX-Exo and Sham-Exo were membrane microcapsules with a size of 40–120 nm (Fig. 2A). Western blot analysis indicated that the exosome markers CD81, HSP70 and TSG101 were highly expressed in OVX-Exo and Sham-Exo but were at low levels or absent in the cell lysates, in contrast to Calnexin (Fig. 2B). In addition, NTA confirmed that OVX-Exo were enriched in the range of 40–200 nm with a peak value of 107 nm (Fig. 2C), which were similar with those in Sham-Exo (Fig. 2D), consistent with previous reports (Kalluri & LeBleu, 2020).

OVX-Exo promoted the expression of osteoclastic markers compared to Sham-Exo

After treatment with M-CSF and RANKL for 4 days, the number of TRAP-positive cells (Figs. 3A and 3B) and the mRNA expression of osteoclastic markers (Fig. 3C), such as tartrate resistant acidic phosphatase (ACP5), CTSK, and NFATc1, were significantly elevated compared with the control group. To explore the effects of Sham-Exo and OVX-Exo on osteoclast differentiation, the exosomes were cocultured with BMMs and the culture medium (complete medium containing M-CSF, RANKL and OVX-Exo or Sham-Exo) was changed every 2 days. After 4 days of coculturing, the mRNA expression of osteoclastic markers were remarkably elevated in the OVX-Exo-treated group compared with the Sham-Exo-treated group (Fig. 3D). In addition, after 24 h of coculturing, confocal microscopy images showed that Sham-Exo and OVX-Exo were incorporated into the cells, while no red fluorescence signal was detected in the cytoplasm of PBS group (Fig. 3E). Which molecule inside the exosomes was at play? We explored this query in the follow-up experiment.

MiRNA expression of OVX-Exo and Sham-Exo

NGS revealed that a total of 708 miRNAs were identified in OVX-Exo and Sham-Exo (Table S2). Among these, 20 miRNAs (14 known and 6 novel) were significantly and differentially expressed (P < 0.05) between OVX-Exo and Sham-Exo (Table 1). The hierarchical clustering illustrated the DE-miRNA profiles (Fig. 4A). The volcano map reflected the distribution of all DE-miRNAs, with rno-miR-27a-3p and rno-miR-196b-5p specially annotated (Fig. 4B).

Target gene prediction and bioinformatics analysis of DE-miRNAs

In this study, 20 DE-miRNAs targeted 5515 mRNAs (Table S3), with rno-miR-27a-3p having the highest number of target genes (1027 genes) (Fig. 5A), which Adcyap1r1 and Bdnf were target genes of 16 DE-miRNAs (Fig. 5B). These target genes were significantly (p < 0.05) enriched in 1200 GO terms (835 biological processes, 161 cellular components, and 204 molecular functions) (Table S4). Fig. 5C lists the 10 most enriched GO terms according to p-value in terms of biological processes, cellular components and molecular functions. KEGG pathway analysis revealed 139 significantly different (P < 0.05) pathways between the OVX-Exo and Sham-Exo (Table S5). The 20 most significantly enriched KEGG pathways of target mRNAs of DE-miRNAs were shown in Fig. 5D. These target genes were enriched in several osteoporosis-related pathways, such as HIF-1, MAPK, Hippo, Ras, and p53 signalings.

MiR-27a-3p and miR-196b-5p promoted osteogenic differentiation of BMSCs

To clarify the regulatory roles of these DE-miRNAs on osteogenic differentiation of BMSCs, we selected miR-27a-3p and miR-196b-5p for further study. According to miRBase (http://www.mirbase.org/), the sequence of human miR-27a-3p is identical to that of rat miR-27a-3p, and human miR-196b-5p shared the same sequence of rat miR-196b-5p. The expression of miR-27a-3p was decreased in BMSCs derived from OVX rats compared to Sham rats (Fig. 6A). During the osteogenic differentiation of BMSCs, the expression of miR-27a-3p was markedly increased at day 5, 7 and 9 after osteogenesis induction (Fig. 6B). The miR-27a-3p was overexpressed by transfection with miR-27a-3p mimic and knocked down with miR-27a-3p inhibitor. After 12 h of transfection, miR-27a-3p mimic- or miR-27a-3p inhibitor-transfected BMSCs showed significantly higher or lower expression of miR-27a-3p, respectively (Fig. 6C). The transfection reagent was then replaced with osteogenic induction solution. After 48 h of induction, protein and mRNA expression of osteogenic differentiation markers, such as ALP, OCN, OSX and RUNX2, were clearly enhanced in BMSCs transfected with miR-27a-3p mimic compared with the mimic-NC group, whereas the miR-27a-3p inhibitor group showed opposite trend (Figs. 6D–6F). Interestingly, miR-196b-5p was studied in the same way and the results were similar to miR-27a-3p (Figs. 6G–6L).

Knockdown of miR-27a-3p and miR-196b-5p promoted osteoclast differentiation

The NGS results were verified by qRT-PCR, indicating that miR-27a-3p and miR-196b-5p were expressed at lower levels in OVX-Exo than that in Sham-Exo (Figs. 7A and 7B). Furthermore, we found that miR-27a-3p and miR-196b-5p expression were decreased during osteoclast differentiation. Briefly, miR-27a-3p and miR-196b-5p expression were decreased at day 4 after osteoclast differentiation induction compared with preinduction (Figs. 7C and 7D). We validated the modulatory effect of miR-27a-3p and miR-196b-5p on osteoclast differentiation by knocking down the expression of miR-27a-3p and miR-196b-5p. The miR-27a-3p inhibitor group had reduced miR-27a-3p expression in osteoclasts (Fig. 7E), while the mRNA expressions of osteoclastic markers ACP5, CTSK and NFATc1 were increased (Fig. 7F). Similarly, the miR-196b-5p inhibitor group had reduced miR-196b-5p expression in osteoclasts (Fig. 7G), while the mRNA expressions of osteoclastic markers were increased (Fig. 7H).