The hBMSCs and HEK-293T cell were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). HEK-293T cell was preserved in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FBS (Gibco) while hBMSCs were maintained in Mesenchymal Stem Cell Basal Medium containing 7% FBS. All mediums were maintained under 37 °C with 5% CO2.
Genechem (Shanghai, China) synthesized the shRNAs against circ_0067680 and negative control sh-NC. Besides, pcDNA3.1 vectors were sub-cloned with CTNNB1 and provided by Genechem. MiR-4429 mimics/inhibitor and negative control were provided by RiboBio (Shanghai, China). Lipofectamine 3000 (Invitrogen) was used for cell transfection. The sequences used have been provided in Additional file 3: Table S1.
Quantitative real-time PCR (RT-qPCR) analysis
Three experiments were implemented. Firstly, total RNA was obtained using TRIzol Reagent (Introgen, Carlsbad, CA, USA), and then reversely transcribed into cDNA using RevertAid First Strand cDNA Synthesis Kit (Thermo fisher, IL, USA). qPCR was performed using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) based on the 2−ΔΔCt method. GAPDH or U6 was internal control. Sequences have been provided in Additional file 3: Table S1.
Cell counting kit-8 (CCK-8)
Three experiments were implemented. Cells were plated in 96-well plates, and treated with 10 μl of CCK-8 solution (Dojindo, Japan). The absorbance was measured at 450 nm.
Alkaline phosphatase (ALP) staining
According to the protocol of NBT/BCIP kit (CoWin Biotech, Beijing, China), ALP staining analysis was carried out. In brief, hBMSCs cells were cultured in OM for 7 days. After fixation and staining, cells were treated with ALP Activity Kit. The experiment was independently conducted in triplicate.
Three experiments were implemented. Via PARIS™ Kit (Ambion, Austin, TX), cytoplasmic and nuclear elements were separated. GAPDH or U6 was respectively regarded as cytoplasmic and nuclear controls.
Fluorescent in situ hybridization (FISH)
hBMSCs cells were permeabilized and fixed, and then hybridized with the RNA FISH probe mix for circ_0067680 in buffer which was synthesized by RiboBio. Hoechst solution was used to dye nuclei. The experiment was independently conducted in triplicate.
RNA pull down assay
Three experiments were implemented. The biotinylated circ_0067680 probe or miR-4429 probe was used to treat hBMSCs. Magnetic beads were then added into cells. After collection and purification, RT-qPCR analysis was implemented.
RNA immunoprecipitation (RIP)
Three experiments were implemented via a Z-Magna RIPTM RNA-binding Protein Immunoprecipitation kit (Millipore Corporation, USA). Cell lysates were treated with Anti-Ago2 (Abcam) antibody and anti-IgG (Abcam) antibody, and analyzed by RT-qPCR.
Luciferase reporter assay
Three experiments were implemented. The sequence of circ_0067680 or CTNNB1 mRNA 3’-untranslated region (3’-UTR) containing wild-type (Wt) and mutant type (Mut) of miR-4429 was inserted into pmirGLO dual-luciferase vector to form pmirGLO-circ_0067680 Wt/Mut and pmirGLO-CTNNB1 3’-UTR-Wt/Mut respectively. Later, miR-4429 mimics and control mimics were separately co-transfected with the reporter gene into hBMSCs cells. After 48 h, the Dual-Luciferase Reporter Gene Assay Kit (Yeasen, Shanghai, China) was applied to measure luciferase activity.
TOP/FOP flash assay
After co-transfection with sh‐NC or sh-circ_0067680#1/2, Wnt/β-catenin report vector TOP/FOP was co-transfected into hBMSCs cells. Relative luciferase activities were detected with a dual-luciferase reporter assay Kit (Promega, Madison, WI, USA). The assay was independently carried out in triplicate.
Western blot analysis
Three experiments were implemented. Separated protein samples were transferred to PVDF membranes (Millipore, Billerica, MA, USA) which subsequently went through incubation with the following primary antibodies against nuclear β-catenin (Abcam, ab32572), p65 (Abcam, ab32536), p-p65 (Abcam, ab76302), AKT (Abcam, ab8805), p-AKT (Abcam, ab38449), c-myc (Abcam, ab32072), cyclin D1 (Abcam, ab16663), RUNX2 (Abcam, ab192256), OSX (Abcam, ab209484), OCN (Abcam, ab93876), C3orf58 (Invitrogen, PA5-26926), Histone H3 (Abcam, ab1791) and GAPDH (Abcam, ab181602) after being blocked with skimmed milk. Afterwards, the blots were incubated with secondary antibody. At last, Chemiluminescence system (GE Healthcare, Chicago, USA) was applied to quantify proteins.
Experimental data were analyzed by SPSS 22.0 statistical software and expressed as mean ± standard deviation (SD). The differences were analyzed with the employment of Student’s t-test or ANOVA. All experiments were independently performed in triplicate. Data was significant when P < 0.05.