BM-MSC culture and characterization

Human bone marrow mesenchymal stem cells (BM-MSCs) were obtained from Procell (Wuhan, China) and cultured in Dulbecco´s Modified Eagle´s Medium (DMEM, Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS, Gibco) and 100 U/mL penicillin (Sigma, St Louis, MO, USA). Only BM-MSCs from passages 3–5 were used for this study.

Flow cytometry was used to characterize BM-MSCs using antibodies against CD45, CD14, CD90, CD73, HLA-DR and CD105 (BD, Biosciences, San Jose, CA, USA).

Cell transfection

Based on previous studies, cell transfections were performed (Maak et al. 2021). Briefly, BM-MSCs were seeded 24 h prior to transfection. sh-IGF2BP3-1-4#, sh-KDM1A-1-4#, sh-circ_AFF4-1-4# or control shRNA were obtained from Genesee Biotech and were inserted into GV102 vectors and constructed by Genepharma (Shanghai, China). These vectors were transfected into BM-MSCs cells (5 × 106 cells per well) using Lipofectamine 2000 (Invitrogen, Invitrogen, CA, USA) in 20 µL lipofectamine. After 6 h, the culture medium was changed to the normal medium. After 48 h, cells were used for the in vitro experiments. For lentivirus infection, lentiviruses carrying sh-KDM1A, circ_AFF4 overexpression lentiviruses, FNDC5 overexpression lentiviruses and correspondence control vectors were obtained from Genepharma and were used to infect BM-MSCs. Cells were selected with 2.5 µg/mL puromycin after 2 days post-transduction. 4–5 days later, puromycin was removed and cells were continuously cultured in the normal medium until fully recovered for animal studies.

Osteogenic differentiation induction

Osteogenic Differentiation Medium BulletKit (Lonza, Basel, Switzerland) was used to induce BM-MSC osteogenic differentiation followed by manufacturer instructions. In brief, 1 × 104 cells/well of BM-MSCs were seeded onto a 24-well plate until reach an 80% confluence. After complete removal of the medium, 1 mL/well of osteogenic medium (OM) was added. Cells cultured in normal DMEM were utilized as controls. The medium was changed every 3 days until 14 days. The cells were harvested for further analyses.

In vivo bone formation study

Adult C57BL/6J male mice that were between 12 and 14 weeks and weighing 25–30 g were given by Hunan SJA laboratory animal Co., Ltd (Changsha, China) and were kept in an animal facility (temperature 22 ± 3 °C, humidity 55% ± 10%, and 12 h lights on/off cycles). The Ethics Committee of the First Affiliated Hospital of Nanhua University (registration number: ChiCTR1900021723) has approved all the experiments.

Sixty mice in total were separated into six groups, (1) control, (2) sh-KDM1A, (3) circ_AFF4, (4) shKDM1A + circ_AFF4, (5) FNDC5, and (6) shKDM1A + FNDC5. BM-MSCs that infected with sh-NC and lentiviruses, sh-KDM1A, circ_AFF4 overexpression lentiviruses, sh-KDM1A plus circ_AFF4 overexpression lentiviruses, FNDC5 overexpression lentiviruses and sh-KDM1A plus FNDC5 overexpression lentiviruses cultured in OM for 1 week and seeded on bioceramic scaffolds with β-tricalcium phosphate and hydroxyapatite mixture (National Engineering Research Center for Biomaterials, Sichuan University, China) based on a previously published protocol (Liu et al. 2021). The BM-MSC-seeded scaffolds were subcutaneously placed on the dorsal side of the mice. After two months, the mice were sacrificed. Femur bone samples were harvested for the following analyses.

Alkaline phosphatase (ALP) staining

ALP staining assay was carried out utilizing an ALP staining kit (GeFan Biotechnology, Shanghai, China). In brief, after being washed with PBS three times, cells were fixed with a fixing solution. Afterward, cells were incubated with ALP staining solution for 30 min in the dark followed by washing with PBS 3 times and visualized with a light microscope (Zeiss, Jena, Germany).

Alizarin red S (ARS) staining

After being fixed with 4% PFA, cells were subsequently incubated with Alizarin Red S solution that was freshly prepared (Sigma-Aldrich) for about 30 min at room temperature. After 3 times wash with PBS the staining was imaged with a microscope (Zeiss).

Actinomycin D and R treatment

BM-MSCs were seeded into six-well plates and maintained until they reach 60% confluency. Afterward, Actinomycin D (5 µg/mL) or DMSO (Sigma-Aldrich) was used to treat cells for 0, 12 and 24 h and then collected. 2 µg of total RNA was treated with RNase R (3 U/µg, Epicentre Technologies, Madison, WI, USA) for 15 min. After treatment, the RNA expression levels were analyzed by RT-qPCR.

RNA fluorescence in situ hybridization (FISH)

After being washed 3 times with PBS, cells were treated with RNase R (Sigma-Aldrich) for 15 min followed by fixation with 4% PFA. The cells were placed onto glass slides and then dehydrated with ethanol. After hybridization, the slides were cleaned three times in 50% formamide/2 × SSC fand and incubated with circ_AFF4 and IGF2BP3 specific probes (Merck Millipore, Billerica, MA, USA) at 37 °C in standard hybridization buffer (900 mM NaCl, 20 mM Tris/HCl, 0.01% sodium dodecyl sulfate and 40% formamide). Afterward, cells treated with the DAPI (Thermo Fisher Scientific, Waltham, USA) for 15 min. The images were taken utilizing fluorescence microscopy with a 60X objective (AXIO, Zeiss).

Immunofluorescent staining

The cells were fixed with 4% PFA for about 15 min followed by blocked with the goat serum and incubated with primary antibodies against Anti-RUNX2 (ab192256, Abcam, Cambridge, UK) at 4 °C overnight. After the secondary antibody (ab150077, Abcam) incubation, the cells were further incubated with DAPI. The images were captured under a confocal microscope (LSM710, Zeiss).

Target prediction

The online bioinformatic tools, BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi), was utilized to predict the binding sequence of circ_AFF4 with FNDC5, and AnimalTFDB (http://bioinfo.life.hust.edu.cn/AnimalTFDB/) was utilized to predict the interaction of KDM1A with circ_AFF4 and FNDC5.

RNA pull-down

Biotinylated IGF2BP3 and the mutants with the mutated putative binding sites for circ_AFF4 were generated by in vitro transcription using TranscriptAid T7 High Yield Transcription Kit (Thermo Scientific) and purified utilizing GeneJET RNA Purification Kit (Thermo Scientific). The cell lysates that were extracted from BM-MSCs were treated with purified IGF2BP3 or mutants and with streptavidin-coated magnetic beads (Invitrogen) in order to pull down the biotin-coupled RNA complex following the user´s instructions. Magnetic beads were washed extensively. The enrichment of circ_AFF4 or IGF2BP3 was examined by RT-qPCR analysis. The bound proteins were eluted and analyzed by SDS-PAGE. The proteins were analyzed by western blot.

RNA immunoprecipitation (RIP) assay

BM-MSCs were collected, and RIP assay was carried out utilizing an EZ-Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Merck Millipore). Briefly, RIP lysis buffer was added to the cells that centrifuged at 2500 g for 10 min and then 13,000 g for 10 min. The pellets were incubated with magnetic beads conjugated to either human anti-IGF2BP3 antibody (ab225697, 1:50, Abcam), or the nonspecific anti-IgG antibody (ab172730, 1:50, Millipore) at 4 °C overnight. Afterward, protein K buffer was added for another 2 h at 4 °C. After centrifuging five times, the co-immunoprecipitated RNA was isolated and used for further RT-qPCR analysis.

Enzyme-linked immunosorbent assay (ELISA) assay

The Irisin levels in BM-MSCs were measured using ELISA kits (NBP3-08117, Novus Biologicals, Littleton, CO, USA) based on the manufacturer’s protocol.

Electrophoretic mobility shift assay (EMSA)

RNA EMSA assay was performed utilizing a LightShift™ Chemiluminescent RNA EMSA Kit (Thermo Fisher Scientific), based on the users´ instructions. In brief, a biotin-labeled oligonucleotide probe was purchased from Sangon Biotech (Shanghai, China) and was used to treat purified protein in a binding buffer for about 30 min. Electrophoresis on 6% nondenaturing polyacrylamide gel in 0.5× TBE buffer was used to resolve the complexes at 100 V for 1 h, which was then transferred to a nylon membrane for 30 min at 400 mA. The membrane was crosslinked, blocked and incubated with HRP-linked streptavidin for 15 min. For supershipft assay, recombinant GST-IGF2BP3 proteins were preincubated with ant-IGF2BP3 antibody (14642-1-AP, Proteintech, Chicago, IL, USA) for 20 min at 0 °C and then incubated with labeled probe for supershift assays.

Dual-luciferase reporter assay

The assay was performed based on a reported protocol (Chen et al. 2020). Briefly, the FNDC5 wild-type sequence (FNDC5-WT) that contains the binding site of circ_AFF4 and the FNDC5 mutant sequence (FNDC5-MUT) were amplified and subsequently cloned into a psiCHECK2 vector (Promega, Shanghai, China). The mutant constructs were generated utilizing a QuickChange site-directed mutagenesis kit based on the user´s manual (Stratagene, CA, USA). Afterward, the BM-MSCs cells were co-transfected with FNDC5-WT, FNDC5-MUT and sh-circ_AFF4#1, sh_circ_AFF4#2, or lentivirus circ_AFF4 or correspondence negative controls, utilizing Lipofectamine 2000 transfection reagent (Invitrogen). The Dual-Luciferase Reporter Assay Kit (Promega) was utilized to examine the luciferase activities following 48 h incubation.

Real-time polymerase chain reaction (RT-qPCR)

The total RNA was extracted from cells or tissues utilizing the Trizol reagent (Beyotime, Shanghai, China). 2 µg of total RNA was reversely transcribed utilizing SYBR Premix Ex Taq (Takara, Dalian, China) based on the manufacturer’s instructions. qPCR was carried out utilizing Taqman® Universal PCR mixture Kit (Thermo Fisher Scientific) based on the user’s instruction. The relative mRNA levels were quantified using the 2−ΔΔCt method and GAPDH was utilized as the housekeeping gene. Primer sequences were purchased from Sangon Biotech (Shanghai, China) and their sequences were listed in Table 1.

Table 1 The primers in qRT-PCR Histological analysis

Femur bone tissue from mice was fixed in 4% PFA, subsequently embedded in paraffin and cut into 5 μm sections. As previously reported (Fischer et al. 2008), the sections were stained with hematoxylin & eosin (H&E). In brief, the sections were incubated with hematoxylin (Sigma-Aldrich) for 3 min and washed for 1 min. Afterward, these sections were stained with eosin (Sigma-Aldrich) for 45s. The sections were visualized utilizing a microscope after dehydrating and mounting.

Immunohistochemistry (IHC) staining

Bone tissues were fixed in 4% PFA and were subsequently embedded in paraffin. IHC staining was performed on 5 μm slides from embedded blocks. Slides were stained using the protocol as described previously (Koukourakis et al. 2005). The sections were incubated with a RUNX2 primary antibody (ab236639, Abcam), for 1 h at RT and followed by incubation with a secondary antibody (ab150077, Abcam). The sections were counterstained with hematoxylin and visualized under a Zeiss confocal microscope.

Masson staining

Trichrome Stain Kit (Sigma-Aldrich) was used for Masson staining based on the manufacturer’s protocol. The images were taken using a light microscope.

Western blot

Total cellular protein was isolated from the cells using the radio-immunoprecipitation assay (RIPA) buffer (Beyotime) containing protease inhibitors at 4 ºC for 30 min. Protein concentrations were measured with BCA protein, separated by SDS-PAGE (30 µg/well), and transferred to polyvinylidene fluoride membranes and blocked. The membranes were incubated with primary antibodies including ALP (ab229126, Abcam), OPN (ab283656, Abcam), RUNX2 (ab23981, Abcam), Colla1 (ab260043, Abcam), and KDM1A (ab62582, Abcam). Afterward, these membranes were washed and followed by incubation with a secondary antibody (ab7090, Abcam). Eventually, an ECL kit (Millipore) was utilized to visualize the protein bands.

Chromatin immunoprecipitation (ChIP)

The EZ-ChIP kit (Upstate, Lake Placid, NY, USA) was used to perform the ChIP assay based on a previously published protocol (Milne et al. 2009). In brief, BM-MSCs were resuspended in SDS lysis buffer and then sheared by sonication. The chromatin fragments were immunoprecipitated with antibodies against KDM1A (ab129195, Abcam), H3K9me3 (ab8898, Abcam) and the purified DNA was analyzed using RT-qPCR.

Statistical analysis

All statistical analysis was performed using GraphPad Prism 5.0. Two groups were compared using an unpaired student t-test. One-way analysis of variance (ANOVA) followed by the Turkey post hoc test was applied in cases of comparison among multiple groups. P < 0.05 was considered significant. All data are shown as mean ± standard deviation. At least three independent experiments were carried out.