Cell culture and osteogenic induction. Mouse bone marrow stem cells (BMSCs; Cyagen Biosciences Inc., Guangzhou, China) were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at 37℃ with 5% CO2 in a humidified chamber. Differentiation of BMSCs were induced in DMEM medium containing 50 mg/mL ascorbic acid (Sigma), 10 mM β-glycerophosphate (Sigma, St. Louis, USA) and 100 nM dexamethasone (Sigma).
Library construction and high-throughput sequencing. RNA sequencing analysis was carried out using the protocol described elsewhere [22]. Briefly, RNA was extracted and converted to double-stranded cDNA. PCR products amplified from those purified cDNA products were purified and treated with terminal repair and ligation primers using NEBNext® UltraTM RNA Library Prep Kit. Then libraries were sequenced using an Illumina HiSeq2000 platform with pair-ended 150 base reads.
Bioinformatics analyses. The prediction is the potential miRNAs that bound to mmu_circ_009056,
online analysis tools RegRNA 2.0 (regrna2.mbc.nctu.edu.tw/detection.html), miRWalk 2.0 (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/) were used.
Transfection of small interfering RNAs (siRNAs), mimics, and inhibitors. The siRNAs of mmu_circ_009056, miR-139-3p mimic, and inhibitor were designed and synthesized by Guangzhou RiboBio Co., Ltd. (Table 1). BMSCs were seeded in 6-well plates at a density of 2×105/well. 50 nM siRNA mimics or inhibitors were transfected into BMSCs sing GenMute™ siRNA Transfection kit and Transfection Buffer (SignaGen Laboratories).
Alizarin Red‑S (ARS) staining assay. Mineralization of nodules was assessed by ARS staining. 2.8×104 per well cells were seeded in 48-well plates. BMSCs were transfected with either 50 nm siRNA1 against mmu_circ_009056 or negative control. Osteoinduction medium was replaced every 2 days. The cells were washed with PBS after 21 days culture and fixed in 4%. paraformaldehyde (Sigma, USA). After 20 min, the cells were washed with deionized H2O and stained with ARS solution (BestBio, Inc.) for 10 min. Mineralization nodules were observed under a microscope (Leica Microsystems GmbH).
Cells were seeded in 48-well plates at a density of 2.8×104 cells/well. BMSCs were transfected with either 50 nm siRNA1 against mmu_circ_009056 or negative control. The medium was changed every 2 days. Following culture for 21 days, cells were washed with PBS and fixed in 4% paraformaldehyde (Sigma, USA). After 20 min, the cells were washed three times with deionized H2O and incubated with ARS solution (BestBio, Inc.) at room temperature for 10 min. The stained cells were observed under a microscope (Leica Microsystems GmbH).
Reverse transcription-quantitative polymerase chain reaction (RT-PCR). Total RNA was isolated from BMSCs using TRIzol reagent (Thermo Fisher Scientific, Inc.). The quantity and quality of RNA were determined with a NanoDrop 2000. Total RNA was converted to complementary DNA (cDNA) by reverse transcriptase (SuperScript III; Invitrogen; Thermo Fisher Scientific, Inc.), using the manufacturer’s recommendation protocol. PCR amplification was performed using gene-specific PCR primers and Taq polymerase (SYBR Premix EX Taq; Takara Bio, Inc.). The primers used in the study are listed in Table I. cDNA amplifications were carried out under thermal cycling (95˚C for 30 sec, then 95˚C for 5 sec, 55˚C for 30 sec, 72˚C for 30 sec for 40 cycles). A cycle quantification (Cq) value was obtained from the Bio-Rad CFX manager software, change in gene expression relative to housekeeping genes between groups was calculated using the 2–ΔΔT method with normalization to GAPDH as previously described [23].
Western blotting. BMSCs were lysed in lysis buffer on ice, and total proteins were extracted using phenylmethanesulfonyl fluoride and radioimmunoprecipitation assay buffer. Protein concentration was determined using a BCA protein Assay Kit (BestBio, Inc.). Total proteins (20 µg) were separated by electrophoresis on a 10% SDS-PAGE gel. Separated proteins were electro-transferred to polyvinylidene difluoride membranes, then the membranes were blocked with 5% skimmed milk. Membranes were then incubated in blocking buffer containing primary antibodies against RUNX2 (1:1,000; Abcam), ALP (1:1,000; Abcam), PAX5 (1:1,000; Abcam) and β-actin (1:5,000; Abcam) overnight at 4˚C. Then the membranes were washed with 0.1% Tween PBS-T and incubated with goat anti-mouse horseradish peroxidase-conjugated secondary antibody (1:5,000; Abcam) for 1 h at room temperature. The membranes were washed with 0.1% Tween PBS-T again and stained with ECL kit (Beijing Dingguo Changsheng Biotechnology Co., Ltd.).
Statistical analysis. Data were presented as the mean values ± standard deviation (means ± SD). The data was analyzed using Student t-test or one-factor analysis of variance (ANOVA) according to the nature of the data. P<0.05 was considered to indicate a statistically significant difference.