Circular RNA circStag1 promotes bone regeneration by interacting with HuR

BMSC isolation and culture

All animal experiments were approved by the Animal Ethics Committee of Jinan University. Sprague-Dawley rats (female, eight weeks old) were obtained from the Medical Experimental Animal Center of Guangdong Province, randomly split into two groups, and then underwent sham operation (n = 10) and bilateral ovariectomy (n = 10) (Fig. S1a, b). Each rat received an intramuscular penicillin injection (80 000 units/rat) for three days after the operation. All rats were euthanized two months after the operation. The bone marrow of each rat was collected from bilateral femurs in an aseptic environment and separated by density gradient centrifugation at a speed of 1 200 r·min−1. Then, BMSCs were cultured in alpha-modified Eagle’s medium (Gibco, Thermo Scientific, USA) with 10% fetal bovine serum (FBS, Gibco, Thermo Scientific, USA) and 1% penicillin and streptomycin (Gibco, USA) at 37 °C with 5% CO2 and 95% humidity (Fig. S1c). The culture medium was replaced every three days. The expression of specific cell surface markers of BMSCs was verified (Fig. S1d)51.

In addition, human BMSCs were purchased from the American Type Culture Collection (Manassas, USA) and cultured in alpha-modified Eagle’s medium (10% FBS and 1% penicillin and streptomycin) at 37 °C with 5% CO2 and 95% humidity.

Human samples

A total of 20 postmenopausal women who underwent spinal fusion surgery at Shenzhen People’s Hospital were included in this study (Table S2). Their bone mineral density was measured and is presented as a T-score. Participants with T scores ≤ −2.5 SD were assigned to the osteoporosis group (n = 10), while those with T scores ≥ −1.0 SD formed the control group (n = 10)13. Participants with other metabolic diseases, such as osteonecrosis, rheumatoid arthritis, and diabetes, were excluded from the study. Bone tissue (100 mg) was obtained from each patient during the operation and then placed in a liquid nitrogen jar for preservation. The sample collection procedures were approved by the research ethics committee of Shenzhen People’s Hospital. Written informed consent was obtained from each participant.

RNA sequencing

The TRIzol method (Invitrogen, USA) was performed to extract the total RNA of BMSCs from the OVX and sham-treated rats (n = 3). Then, the circRNA expression profile in BMSCs was detected by RNA sequencing (RiboBio Co., Ltd., Guangzhou, China)52. In brief, rRNA and linear RNA were first removed by using the RiboBio rRNA removal kit and RNase R. Then, circRNA was reverse transcribed to cDNA. High-throughput sequencing was performed to generate the raw data by using an Illumina HiSeq 3000 system. Then, CIRI2 and CIRCexplorer2 software were used to identify circRNAs. Subsequently, sequence prediction, expression value calculation, and expression difference analysis were performed for the identified circRNAs. Any circRNA with P < 0.05 and fold change ≥2 was regarded as a differentially expressed circRNA20. The expression trends of these dysregulated circRNAs are shown by heatmaps and volcano plots. Pathway enrichment among the host genes of the dysregulated circRNAs was identified using KEGG pathway enrichment analysis.

Quantitative real-time PCR

AG RNAex Pro Reagent (AG21102, Accurate Biotechnology, Hunan, China) was used to collect total RNA from bone samples or BMSCs following the standard protocol. Then, total RNAs were reverse transcribed to cDNA using the PrimeScript RT reagent Kit (TaKaRa, Tokyo, Japan). Sangon Biotech Co., Ltd., (Shanghai, China) designed and synthesized the primers for quantitative real-time PCR (qPCR) (Table S3). Then, qPCR was performed using TB Green Premix Ex Taq (TaKaRa, Tokyo, Japan) on a StepOne Plus quantitative PCR system (Applied Biosystems, Foster City, USA). Glyceraldehyde-3-phosphate dehydrogenase mRNA (Gapdh) was used as the internal control for circRNAs and mRNAs, while U6 was used as the internal control for miRNAs.

CircStag1-HuR modeling structure

The potential interacting proteins of circStag1 were predicted using RBPDB (http://rbpdb.ccbr.utoronto.ca/) and the Circular RNA Interactome (https://circinteractome.nia.nih.gov/) databases. For circRNA structure modeling, we used RNAComposer (http://rnacomposer.cs.put.poznan.pl/) to predict the three-dimensional structure of circStag1, running it in interactive mode with the circStag1 sequence as input53,54. For this analysis, we chose the select secondary structure prediction method, and we chose the ContextFold method from the drop-down list55. For the HuR protein structure, we separately chose the HuR-RRM1-2 structure (Protein Data Bank code 4EGL) and HuR-RRM3 structure (Protein Data Bank code 6GC5) as the docking template56.

The HADDOCK2.4 webserver (https://bianca.science.uu.nl/haddock2.4) was used to separately dock HuR-RRM1-2 and HuR-RRM3 with circStag and predict the biomolecular interactions between circStag1 and the RNA recognition motifs of the HuR protein57. The input data consisted of the circStag1 and HuR models described above; ambiguous restraints for the complex docking interaction interface and the unambiguous distance restraints were used.

Cell transfection

Small interfering RNAs against circStag1 and HuR (Table S4), control siRNAs, overexpression plasmids for HuR (ov-HuR) and circStag1 (ov-circStag1), and mutant forms of ov-circStag1 were synthesized by Hanbio Co., Ltd. (Shanghai, China). BMSCs were seeded into plates for 24 h and then transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the standard protocol.

Western blot

Total protein was extracted with the Total Protein Extraction Kit (Signalway Antibody LLC, Maryland, USA) following the manufacturer’s instructions. Proteins were separated on SDS-PAGE gels and then transferred to PVDF membranes (Millipore, Massachusetts, USA), which were blocked with TBST containing 5% skim milk and incubated with anti-Stag1 (Proteintech, China; 14015-1-AP; 1:1 000), anti-β-catenin (Santa Cruz Biotechnology, USA; sc-7963; 1:1 000), anti-Lrp5 (Thermo, USA; 36-5400; 1:1 000), anti-Lrp6 (Thermo, USA; PA5-101047; 1:1 000), anti-HuR (Abcam, UK, ab200342; 1:1 000), anti-β-tubulin (Cell Signaling Technology, USA; 2146; 1:1 000), anti-Alp (Santa Cruz Biotechnology, USA; sc-365765; 1:1 000), anti-Opn (Santa Cruz Biotechnology, USA; sc-21742; 1:1 000), anti-Ocn (Santa Cruz Biotechnology, USA; sc-390877; 1:1 000), anti-Lamin B1 (Abcam, UK; ab16048; 1:5 000), and anti-Gapdh (Abcam, UK; ab9485; 1:5 000) at 4 °C overnight. Finally, the membranes were hybridized with secondary antibodies at room temperature for 1 h and visualized using RapidStep™ ECL Reagent (Millipore, USA).

Immunofluorescence

Samples were fixed in 4% paraformaldehyde, permeabilized with 0.25% Triton X-100 in PBS for 15 min, blocked with 10% goat serum for 1 h at 37 °C and incubated with anti-Lrp5, anti-Lrp6, anti-β-catenin, anti-HuR, anti-Alp, anti-Opn, and anti-Ocn antibodies overnight at 4 °C. Next, the samples were incubated with the secondary antibody in a 1:200 dilution ratio in PBS for 30 min at 37 °C in the dark, followed by nuclear staining with DAPI (Abcam, Cambridge, UK). Fluorescence images were acquired using a Leica DMi8 microscope (Carl Zeiss MicroImaging GHBH, Jena, Germany).

RNA fluorescence in situ hybridization

Cy3-labeled probes for the backsplicing junction of circStag1 (Table S5), U6, and 18S were synthesized by RiboBio (RiboBio Biotechnology, Guangzhou, China). The hybridization was performed in a humidification chamber at 37 °C for 16 h. The circStag1 signals were detected using a Fluorescent in Situ Hybridization Kit (RiboBio, Guangzhou, China) following the manufacturer’s instructions41. Fluorescence images were collected using a Leica DMi8 microscope.

Biotin-labeled RNA pulldown

CircStag1 probes with biotin labels and control probes were synthesized by RiboBio Biotechnology (Table S5). Biotin-labeled RNA pulldown was performed using the Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo, USA) following the manufacturer’s instructions. Briefly, the probes (50 pmol·L−1) were incubated with streptavidin-coated magnetic beads for 30 min at room temperature. The cells were lysed with Thermo Scientific Pierce IP Lysis Buffer (Thermo, USA), incubated with the probe-bound magnetic beads and rotated for 1 h at 4 °C. Then, the probe-bead-protein complex was washed three times with washing buffer. The proteins in the complex were extracted and detected by western blotting.

RNA immunoprecipitation

A Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) was used to validate the interaction between circStag1 and HuR protein. In brief, magnetic beads were coated with 5 μg of antibodies, including anti-HuR (Abcam, UK) and anti-immunoglobulin G (IgG) (Millipore, USA), for 30 min at room temperature. The cell lysate from 2 × 107 cells was incubated with antibody-coated magnetic beads overnight at 4 °C. Then, the bead-protein-RNA complexes were washed with RIP washing buffer six times, incubated with proteinase K digestion buffer, and rotated for 30 min at 55 °C. Finally, RNA was extracted and reverse transcribed to cDNA, and the level of circStag1 was detected via qPCR and normalized to the input.

Osteogenic induction

BMSCs were seeded into 6- or 48-well plates and cultured in alpha-modified Eagle’s medium. After cell attachment, the culture medium was changed to osteogenic medium supplemented with 10% FBS, 50 μg·mL−1 ascorbate, 10 μmol·L−1 β-glycerophosphate, 0.1 μmol·L−1 dexamethasone, and 10 μmol·L−1glutamine (Cyagen Biosciences, Guangzhou, China). The osteogenic induction medium was replaced every 2 days.

Alizarin red and ALP staining

The osteogenic differentiation capability of BMSCs was validated by Alizarin red S staining (ARS) and ALP staining at Day 7 after transfection. In brief, BMSCs were seeded in 48-well plates and cultured in osteogenic medium. After 7 days of induction, the cells were fixed with 4% paraformaldehyde for 10 min and stained with ARS staining solution (Cyagen, China) at pH 8.3 for 30 min. After two washes, the staining area was photographed by light microscopy (TS2-S-SM, Nikon, Japan), and the positive area was quantified with ImageJ software. Additionally, a BCIP/NBT ALP Color Development Kit (Beyotime, China) was applied to detect the expression of Alp. The stained cells were also photographed by light microscopy.

CircStag1-AAV construction and injection

Adeno-associated virus (AAV) was used as a vehicle for circStag1 to validate its in vivo function58. In brief, recombinant AAV-9 containing the circStag1 sequence (circStag1-AAV) was synthesized by Hanbio Co., Ltd., (Shanghai, China) and diluted in PBS to 1 × 1013 virus particles per mL. Female Sprague-Dawley rats (eight weeks old) were purchased and assigned to the sham control group, OVX group, control AAV-injected OVX group, and circStag1-AAV-injected OVX group (n = 6). Three days after ovariectomy, circStag1-AAV (or the same dose of normal saline or control-AAV) was injected into the tail vein of the rats at a dose of 2 × 1012 vg/rat per week for 4 weeks59. Calcein green (10 mg·kg−1 body weight) was injected intraperitoneally at Days 9 and 2 before euthanasia13. One week after the last AAV injection, all rats were euthanized, and the samples of primary organs of each rat were collected to test the impact of circStag1-AAV on primary organs. The femurs and tibias were collected for detecting bone formation and mechanical strength analysis.

Micro-CT

The right femurs (fixed in 4% paraformaldehyde) were scanned by using a micro-CT system (SkyScan 1276, Bruker, Belgium). The parameters used in micro-CT scanning were as follows: current of 100 μA, voltage of 80 kVp, pixel size of 20 µm, and exposure time of 926 ms. After scanning, NRecon software was used to construct 3D images of the distal metaphysis. Parameters of bone static histomorphometry, including BMD, Tb.N, BV/TV, and Tb.Th, were analyzed.

Bone histomorphometry

The trabecular bone of the distal right femur was dehydrated with a 20% sucrose solution, and frontal sections (10 µm thickness) were made using a Leica SM2500E microtome (Leica Microsystems). Then, fluorescence microscopy was used to collect fluorescent images of calcein green. Then, the dynamic bone histomorphometric parameters, including MAR and BFR/BS, were calculated using ImageJ (NIH, USA).

Bone mechanical properties

Three-point bending assays were conducted to examine the mechanical properties of rat tibias using a strength testing device (AG-IS, Shimadzu, Japan). The fresh tibias were wrapped in normal saline-soaked gauze and then centered longitudinally with the anterior surface on the two lower support points (20 mm distance). A third rounded bar was placed at the medial surface of the diaphysis with a 10 N preload strength. Then, a constant displacement rate of 2 mm·min−1 was applied until fracture occurred during three-point bending (Trapezium X, Shimadzu, Japan). The mechanical data were generated and analyzed to determine the maximum load, stiffness, and maximum strength60.

Statistical analysis

The expression data are presented as the mean ± standard deviation (x ± s). GraphPad Prism 7.5 was used to analyze the differences. The difference between the two groups was analyzed using Student’s t test. One-way analysis of variance with Tukey’s test as the post hoc test was carried out to compare the differences among three or more groups. P < 0.05 was considered statistically significant. The differentially expressed circRNAs were those with P < 0.05 and fold change ≥2. The Pearson correlation coefficient was used to analyze the correlation between circStag1 and its downstream genes.

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