Construction of Ythdf1 knockout mice
This study was approved by the Institutional Ethics Committee of Xinhua Hospital affiliated to Shanghai Jiaotong University School of Medicine (XHEC-NSFC-2019-153). All applicable institutional and national guidelines for the care and use of animals were followed. Ythdf1 CRISPR–Cas9 knockout (Ythdf1 KO) mice were constructed as previously reported [31], the KO mice were gifts from the School of Life Science and Technology, Shanghai Tech University. The genotype of all progeny was confirmed by PCR analysis of the DNA extracted from tail biopsies. All procedures were performed in accordance with NIH guidelines for animal experimentation. Five animals were housed per cage under standard laboratory conditions (21 ± 2 °C, humidity 60% ± 10%, and a 12 h/12 h dark/light cycle). All the animals used in the present study were male mice with the same C57BL/6 genetic background. The WT and Ythdf1 KO mice from the same litter were matched and were randomly selected for further experiments. For each experiment, animals were transferred to the experimental room 30 min prior to the experiment to acclimate them to the environment.
µCT and histomorphometric analyses
The tibias from WT and Ythdf1 KO mice were skinned and fixed with 4% paraformaldehyde and then scanned using an 80 µCT system (Scanco Medical, Bassersdorf, Switzerland) at a spatial resolution of 5 µm (55 kV, 114 mA, and 500 ms integration time). The proximal metaphysis between 1.5 and 4 mm distal to the growth plate was set as the region of interest. Three-dimensional and two-dimensional images were generated using the CTvol program (Skyscan). Micro-architectural parameters, including BV/TV, Tb.Th, Tb.N, and Tb.Sp, were obtained and analyzed.
To evaluate the BFR in vivo, 12-week-old WT and Ythdf1 KO mice were intraperitoneally injected with green fluorescent calcein (5 mg/kg, Sigma-Aldrich) and Alizarin Red S (40 mg/kg, Sigma-Aldrich) at 3 and 10 days before euthanasia. The tibias were then dissected, fixed, dehydrated, and embedded in methyl methacrylate resin, after which the tissue sections were observed under a laser-scanning microscope (LSM5 PASCAL; Carl Zeis).
Lentiviral Packaging
pLVX-IRES-puro, pLVX-IRES-puro-YTHDF1, pLVX-IRES-puro-Ythdf1, pLVX-IRES-puro-ZNF839, and pLVX-IRES-puro-Zfp839 were transfected into the HEK293T viral packaging cell line together with pSPAX2 and pMD 2.G. Exactly 48 h after transfection, the cells were harvested for RT-PCR or Western blot analysis to verify the packaging efficiency.
The lentiviral knockdown vectors pLKO.1-eGFP for YTHDF1, ZNF839 and Runx2 were constructed, and the virus was prepared according to previous publications. Table 1 lists the target sequences of the shRNAs for YTHDF1, Zfp839, and Runx2.
Immunohistochemical staining
The dissected tibia samples were fixed in 4% polyoxymethylene for 24 h, decalcified in 12.5% ethylenediaminetetraacetic acid (EDTA) (pH = 7.0) for 21 days, and then embedded in paraffin and sectioned for staining. Serial sections (6 µm) of the tibias were incubated with primary rabbit polyclonal anti-Col1a1 (1:500 dilution) overnight at 4 °C.
A horseradish peroxidase–streptavidin detection system (Dako, Glostrup, Sweden) was used to detect immunoreactivity, hematoxylin was used to counterstain the nuclei. Immunohistochemical analysis was performed with a light microscope (Leica, Germany).
Immunofluorescence assay
Paraffin-embedded sections (5 µm thick) of the decalcified tibia from WT and Ythdf1 KO mice were incubated overnight at 4 °C with antibodies (Abs) against Ythdf1 and Zfp839 respectively, and covered with 4’,6-diamidino-2-phenylindole (DAPI) to visualize the nuclei. The primary antibodies were detected using a fluorescein isothiocyanate-conjugated anti-rabbit IgG secondary Ab. The stained sections were observed under a laser confocal microscope (Cell Observer, ZEISS, Germany).
A total of 2 × 105 BMSCs were seeded in a 30-mm confocal dish. After 72 h osteogenic induction, the cells were fixed in 4% paraformaldehyde, permeabilized in 0.2% Triton X-100 and probed with specific primary antibodies for Zfp839 (LSBio, LS-C658560, Seattle, WA, USA) and Runx2 (Abcam, ab76956, Cambridge, UK). The primary Abs were then detected using matching anti-mouse or anti-rabbit IgG secondary Abs. Then cells were also co-stained with DAPI to detect nuclei and observed under a laser confocal microscope (Cell Observer, ZEISS, Germany).
BMSC culture and osteogenic differentiation
To isolate mouse BMSCs, we first extracted bone marrow cells from the femurs and tibias of Ythdf1-KO mice by flushing the cells with a modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (#16000-044; Gibco, AUS), 100 U/mL penicillin G, and 100 mg/L streptomycins (#SV30010; HyClone, Logan, UT, USA). The bones were crushed with a pestle and treated with collagenase (Wako, Osaka, Japan), after which BMSCs were plated on 10 cm culture plates and incubated in 5% CO2 at 37 °C. The medium was changed twice every week to remove unattached cells until confluence was achieved.
hBMSCs were obtained from nine young healthy male individuals (age range: 20–30 years old, average: 26.4 years) who underwent traumatic femoral or tibia shaft fracture treatment by intramedullary nailing and nine aged osteoporotic male patients (age range: 70–80 years old, average: 75.2 years) who received total hip arthroplasty after providing written consent. Cell extraction and passage were performed as previously described [32]. Briefly, bone marrow blood from donors was filtered through a 100 µm nylon mesh cell strainer and then incubated in basal medium (BM) [low glucose Dulbecco’s modified Eagle’s medium (#SH30021.01; HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (#16000-044; Gibco, AUS), 100 U/mL penicillin G, and 100 mg/L streptomycin (#SV30010; HyClone, Logan, UT, USA)] at 37 °C in a humidified atmosphere containing 5% CO2. The established hBMSCs were then used for subsequent experiments at passages 3 to 7.
For osteogenic induction, BMSCs were seeded in six-well plates and cultured in osteogenic medium (BM supplemented with 1 nM dexamethasone (#D4902; Sigma-Aldrich), 50 mM ascorbic acid (#A4403; Sigma-Aldrich), and 20 mM β-glycerolphosphate (#G9891; Sigma-Aldrich). The culture medium was changed every 3 days.
ALP and Alizarin red staining
For ALP staining, cells were washed thrice with phosphate-buffered saline (PBS) and fixed with 4% polyoxymethylene after 7 days of osteogenic induction. Then, the cells were incubated with a 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium staining solution. After a 15 min incubation at 37 °C, the cell layer was washed thrice with deionized water and observed under a digital camera. For Alizarin Red staining, the cells were fixed after 14 days of osteogenic induction with 4% polyoxymethylene for 15 min and stained with Alizarin Red S solution (Sigma-Aldrich) for 15 min until they were orange-red in color. After staining, the cells were washed thrice with deionized water and observed under a digital camera.
Quantitative RT-PCR
Total RNA of cultured cells was extracted using TRIzol reagent (Invitrogen, CA, USA), and cDNA was synthesized with PrimeScript RT Master Mix cDNA Synthesis Kit (Takara, Japan) using 2 µg extracted RNA per sample. RT-PCR was performed with a Roche LC 480 system using SYBR1 Premix (TaKaRa, Inc., Dalian, China) following the manufacturer’s instructions. Relative gene expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the data were analyzed using the Ct (2−ΔΔCt) method. Table 2 lists the primers used in the study.
Western blot
After being washed with ice-cold Dulbecco’s PBS, cells were lysed in RIPA buffer (Biocolors, R0095, Shanghai, China) containing 1% PMSF (Meilunbio, MA0001, Dalian, China). Approximately 20 ug of proteins were resolved on 10% SDS-PAGE gels (Bio-Rad, Richmond, CA) and transferred to polyvinylidene difluoride membranes (Merch, ISEQ00010). The membranes were blocked with Tris-buffered saline containing 5% non-fat milk and 0.1% Tween-20 for 1 h. Then, they were sequentially incubated with primary and secondary antibodies. The immunoblots were visualized using an enhanced chemiluminescence detection system (Millipore, Billerica, MA, USA). The primary antibodies used for Western blot analysis included YTHDF1 (Proteintech,17479-1-AP, Beijing, China), ZNF839 (LSBio, LS-C658560, Seattle, WA, USA), and GAPDH (CST, 5174, Danvers, MA, USA). A horseradish peroxidase-labeled secondary antibody was also used in our work.
Luciferase assay
Briefly, ALP, Osterix, and OCN gene promoter-luciferase reporters; flag-tagged Zfp839; Myc-tagged mouse Runx2; and empty vector pcDNA3.1 plasmid were transfected into HEK-293T cells using Lipofectamine 2000 (Life Technologies, USA). After 24 h of incubation, the cells were washed twice with ice-cold PBS and lysed. Then, luciferase activity was measured using a luciferase reporter assay system (Promega, USA) on a multi-plate reader (Bio-Tek Instruments, USA) in accordance with the manufacturer’s instructions. All data are presented as the mean ± standard deviation (SD) of three independent experiments.
MeRIP-Seq
The m6A RIP was performed with GenSeqTM m6A-MeRIP Kit (GenSeq Inc., China) following the manufacturer’s instructions. Briefly, TRIzol reagent (Invitrogen) was used for the isolation of total RNA from hBMSCs, and random mRNA fragments (−200 nt) were generated by using an RNA fragmentation reagent. Samples were then incubated with anti-m6A-pAb at 4 °C for 2 h. The mixture was immunoprecipitated by incubation with Pierce™ Protein A/G Magnetic Beads at 4 °C for 3 h. Methylated fragments were then eluted from the beads with m6A and precipitated with ethanol after extensive washing. Both the input sample without IP and the m6A IP samples were used for RNA-Seq library generation using the NEBNext® Ultra II Directional RNA Library Prep Kit (New England Biolabs, Inc., USA). The quality of the library was evaluated using a BioAnalyzer 2100 system (Agilent Technologies, Inc., USA), and library sequencing was performed on an Illumina HiSeq instrument with 150 bp paired-end reads.
RIP-Seq
RIP was performed using the EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore) following the manufacturer’s protocol. Briefly, hBMSCs transfected with pLVX-YTHDF1 and the pLVX-Vector were homogenized in lysis buffer supplemented with protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific) and an RNase inhibitor (Promega). The beads were incubated with the YTHDF1 antibody (Proteintech) or normal IgG at room temperature for 30 min and then were incubated with lysate supernatant in IP buffer supplemented with EDTA overnight at 4 °C. After extensive washing with IP buffer, the beads were treated with proteinase K for 30 min at 55 °C with occasional shaking. RNA was purified from the supernatant using TRIzol reagent following the manufacturer’s instructions. rRNA was removed from the immunoprecipitated RNA and input RNA samples (New England Biolabs, Inc., Massachusetts, USA). RNA libraries were constructed using rRNA-depleted RNA with NEBNext® Ultra™ II Directional RNA Library Prep Kit (New England Biolabs, Inc., Massachusetts, USA) following the manufacturer’s instructions. The libraries were controlled for quality and quantified using a BioAnalyzer 2100 system (Agilent Technologies, Inc., USA). Library sequencing was performed on an Illumina HiSeq instrument with 150 bp paired-end reads.
Statistical analysis
All data are presented as the mean ± standard error of the mean. All experiments were conducted with a minimum of three independent biological replicates. Graphs were generated with GraphPad Prism 6.0, and statistical analyses were performed using SPSS software (version 16.0; SPSS, Inc., Chicago, IL). Significant differences were analyzed by unpaired two-tailed Student’s t test for comparison between two groups and one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. All tests were two-sided with a P value of 0.05 as the boundary of statistical significance: *P < 0.05, ***P < 0.01.
