Down-regulation of FTX promotes the differentiation of osteoclasts in osteoporosis through the Notch1 signaling pathway by targeting miR-137

Patients with hip fractures at the third People’s Hospital of Dongguan City were enrolled in this study, who were divided into 2 groups of OP patients and non-OP patients (control group). Osteoporosis were defined according to WHO criteria for classification of osteopenia and osteoporosis [12]. To be included, the fracture of the patients must meet the definition of fragility fracture caused by falling or low energy instead of violence or high-energy traumata. Patients with liver and kidney diseases; osteoarthritis; metabolic bone disease, ovarian diseases or other endocrine diseases were excluded. Also, patients underwent hormone therapy in recent 3 months or other chronic corticosteroid use were excluded due to the influence of these drugs on lncRNA and miRNA expression. Postmenopausal women with OP fractures (n = 40) and healthy volunteers (n = 40) were selected, who received hip replacement surgery in the hospital. Supplementary file 2 showed the patients’ background regarding their age, gender, and BMI. RNA was extracted from the whole bone. According to the T-score of BMD, the patients were divided into two groups: the normal group (T-score ≥ − 1.0) and the OS group (T-score ≤ − 2.5). Serum and bone samples were placed in endoprosthesis. All patients signed informed consent. This research was approved by the Ethics Committee of the third People’s Hospital of Dongguan City.

Peripheral blood mononuclear cells (PBMC) had extraction according to Sorensen et al. [21]. CD14 antibody magnetic cell sorting (Miltenyi, Germany) was utilized to purify CD14 + PBMCs. CD14 + PBMCs were seeded in at 250, 000 cells/well. Cells were growing in alpha minimum essential medium (Invitrogen, USA) with 10% FBS (Gibco, CA), 50 IU/mL penicillin, and 50 mg/mL streptomycin, at 37 °C with humidity and 5% CO₂. The culture medium was added in 25 ng/mL MCSF and 25 ng/mL RANKL (R&D Systems, USA).

4, 000 cells were seeded to a well from 96-well plate and cultured in alpha minimum essential medium with 10% FBS and antibiotics. 10 μL CCK-8 was added. We observed the signals at 2 h after the culture at 450 nm.

Total RNA was extracted by TRIzol (Invitrogen, USA). 0.5 μg RNA was reverse-transcribed by PrimeScript (Takara Bio, Japan). 1 μg cDNA was employed to detect mRNA expressions by qRT-PCR with SYBR Kit (Takara Bio, Japan). GAPDH and U6 were regarded as references. Primers sequences are in the below:

GAPDH (forward: 5′-TGGATTTGGACGCATTGGTC-3′ and reverse: 5′-TTTG.

CACTGGTACGTGTTGAT-3′);

TNFRSF1B (forward: 5′-CGGGCCAACATGCAAAA.

GTC-3′ and reverse: 5′-CAGATGCGGTTCTGTTCCC-3′);

ACP5 (forward: 5′-GACT.

GTGCAGATCCTGGGTG-3′ and reverse: 5′-GGTCAGAGAATACGTCCTCAAA.

G-3′);

MMP-9 (forward: 5′-TGTACCGCTATGGTTACACTCG-3′ and reverse: 5′-GG.

CAGGGACAGTTGCTTCT-3′);

MMP-2 (forward: 5′-TGACTTTCTTGGATCGGGT.

CG-3′ and reverse: 5′-AAGCACCACATCAGATGACTG-3′);

TRAP (forward: 5′-TC.

ACCCTGACCTATGGTGC-3′ and reverse: 5′-GCCGGACTCCAATGTTAAAGC-3′);

cathepsin K (forward: 5′-CTGGCTGGGGTTATGTCTCAA-3′ and reverse: 5′-GGCT.

ACGTCCTTACACACGAG-3′).

FTX-WT (wild-type), FTX-MUT (mutant), Notch1-WT, Notch1-MUT were designed via the clone of WT 3′UTR or MUT 3′UTR to pMir-reporter vector (Ambion, USA). CD14 + PBMCs had co-transfections with luciferase plasmid, miR-137 mimic or miR-NC by Lipofectamine 2000. Dual-luciferase reporter assays (Promega, USA) were used to identify their binding effects.

For pull-down assay, miR-137 without complementary sites with Notch1 was seen as internal reference (termed NC). MiR-137, miR-137-Mut, and NC were labeled with biotin to generate Bio-miR-137, BiomiR-137-Mut, and Bio-NC by GenePharma Company (Shanghai, China). Thereafter, they were transfected into CD14 + PBMCs. Forty-eight hours later, cells were harvested and incubated with Dynabeads M-280 Streptavidin (Invitrogen) for 10 min. After washing three times with buffer solution, the enrichments of bound RNAs were quantified and analyzed by qRT-PCR.

RIP assay was performed using the RNA-Binding Protein Immunoprecipitation Kit (Millipore). Firstly, we lysed CD14 + PBMCs and incubated them with AGO2 antibody or IgG. RNA-protein complexes were immunoprecipitated with protein A agarose beads. Next, we extracted the RNAs using Trizol (Invitrogen, USA). The IP-western was employed to measure AGO2 protein and qRT-PCR was conducted to detect the mRNA expression of FTX, using RIPAb+ Ago2-RIP Validated Antibody and Primer Set, 03–110, Merck Millipore, US.

Induced osteoclasts were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature and then stained by TRAP fluid for 1 h. TRAP solution was prepared by mixing acetate buer (pH 5.0), 50 mM sodium tartrate, naphthol AS-MX phosphate (Sigma Aldrich, Tokyo, Japan), and Fast Red Violet LB salt (Sigma Aldrich, Tokyo, Japan). After removal of the TRAP fluid, the plate was washed three times with phosphate-buffered saline (PBS). TRAP-positive multinuclear cells were observed using an inverted microscope.

Mature osteoclasts were generated by coculturing of mouse bone marrow cells and osteoblasts with VitD3 (10 nM) on collagen gels for 6 days. The generated osteoclasts were replated on an Osteo Assay Surface plate (Corning, NY, USA), allowed to settle for 2 h, and then incubated with different concentrations of WESS for 24 h. After the incubation period, osteoclasts were visualized by TRAP staining. Resorption pits were photographed and analyzed by using Image J software, after removing cells by using sodium hypochlorite bleach.

Protein was separated by 10% SDS-PAGE (Thermo, USA) and sent to PVDF membrane (Millipore, USA). The membrane was blocked by 5% BSA. Next, we treated the membranes with anti-Notch1 (1:1000, Abcam, UK), anti-GAPDH (1:1000, beyotime, China) at 4 °C for a night and then HRP-anti-rabbit (1:1000, beyotime, China) for 2 h at 25 C. We detected the signals using the ECL (Bio-Rad Lab, USA).

After differentiation, cells were collected, washed and suspended. Then, it was incubated by Annexin V at 37 °C for 10 min, and stained by propidium iodide. Flow cytometer was employed to measure cell apoptosis.

The expressions of FTX in serum and bone tissues of OP patients were analyzed. The expressions of FTX in patients’ serum (n = 30) were down-regulated compared with control (n = 21) (Fig. 1a). It is negatively related to the miR-137 expressions (Fig. 1b). In addition, the expressions of FTX in patients’ bone tissues (n = 7) were down-regulated compared with control (non-OP) (Fig. 1c) (n = 7). miR-137 was up-regulated in OP patients’ bone tissues (Fig. 1d).

pcDNA-FTX was efficiently over-expressed successfully in CD14 + PBMCs, according to Fig. 2a. Figure 2b showed that osteoclast marker of TRAP, NFATC1, CTSK, and TRAF6 were hindered by FTX. Figures 2c-e demonstrated that the viabilities of CD14 + PBMCs were decreased by FTX, but cell apoptosis rate was promoted under FTX over-expressions. Figure 2f showed the experiments from TRAP-stained osteoclasts from each group, which revealed that pcDNA-FTX greatly inhibited osteoclast differentiation. It was obvious that FTX regulated osteoclast differentiation in CD14 + PBMCs.

Figure 3a predicted the shared binding sites between FTX and miR-137 using bioinformatics. It also showed the relevant sequences of FTX-WT, miR-137, and FTX-MUT. Figure 3b investigated the successful transfection of miR-137 in cells. Next, we conducted the experiments to study the interactions between miR-137 and FTX in OP by luciferase reporter assays and RIP assays in CD14 + PBMCs. RIP is a powerful method to investigate the physical associations between proteins and RNA molecules. Here, we utilized RIP experiment to verify the binding effect between FTX and miRNA. From Fig. 3c, miR-137 over-expressions resulted in a lower activity of FTX-WT. Figure 3d showed the RIP for binding effects between FTX and miR-137 with the AGO2 antibody. In contrast to the control IgG, higher FTX levels were found in the AGO2 antibody. Figure 3e revealed that over-expressions of FTX could down-regulate miR-137 mRNA expressions in CD14 + PBMCs.

According to Fig. 4a, osteoclast-specific genes of TRAP, NFATC1, CTSK, and TRAF6 were all inhibited by FTX suppression, but this result was blunted by a miR-137 mimic. Figure 4b and c revealed that the viabilities of CD14 + PBMCs were mitigated by FTX but the effects were blunted by a miR-137 mimic. Cell apoptosis was promoted by FTX but the effects were blunted by a miR-137 mimic. Supplementary figure 1A showed the apoptotic rate for early apoptotic cells (LR), and late apoptotic cells (UR). It was noticed that late apoptotic cells appeared under the transfection of pcDNA-FTX. However, this effect was also attenuated by miR-137 (supplementary figure 1B). Figure 4d showed the experiments from TRAP-stained osteoclasts from each group, which revealed that pcDNA-FTX greatly inhibited osteoclast differentiation, while miR-137 mimic attenuated this effect. The data pointed out that FTX regulated osteoclast differentiation in CD14 + PBMCs.

Figure 5a predicted the binding site in Notch1 and miR-137 using bioinformatics. It showed the relevant binding sequences of Notch-WT, miR-137, and Notch-MUT. From Fig. 5b, miR-137 over-expressions resulted in the decreased luciferase activity of Notch1-WT. Figure 5c showed the pull-down assays. We found that Notch1 was pulled-down via bio-miR-137 but not bio-NC or bio-miR-137-Mut. Figure 5d and e demonstrated that over-expressions of miR-137 could down-regulate Notch1 mRNA and protein expressions in CD14 + PBMCs, but over-expressions of FTX could up-regulate Notch1 these expressions.

To further illustrate the mechanisms underlying miR-137 and osteoclast differentiation, we evaluated the relative genes and cells in this process. The osteoclast-specific genes of TRAP, NFATC1, CTSK, and TRAF6 were all inhibited by miR137 suppression, but this result was blunted by shNotch (Fig. 6a). Furthermore, the viabilities of CD14 + PBMCs were mitigated by mirR-137 but the effects were blunted by a miR-137 mimic (Figs. 6b & c). Cell apoptosis was promoted by FTX but the effects were blunted by shNotch. Figure 6d showed the experiments from TRAP-stained osteoclasts from each group, which revealed that mir137 inhibitor greatly inhibited osteoclast differentiation, while shNotch attenuated this effect. Resorption pit assay also showed the same results (Fig. 6e.) The data pointed out that miR-137-notch regulated osteoclast differentiation in CD14 + PBMCs.