Collection of patient samples
This study was approved by the Ethics Committee of the Affiliated Zhujiang Hospital of Southern Medical University. All participants consented to participate in the study. Paired OS and adjacent non-cancerous tissues were collected from biopsies of 55 patients before commencement of neoadjuvant chemotherapy or radiotherapy. The histological diagnosis of OS was performed independently by two experienced pathologists. Upon extraction, the tissues were immediately frozen in liquid nitrogen and kept at -80 °C. Table 1 shows the patients’ clinical characteristics.
Cell culture
Human osteoblast cell line (hFOB1.19) and OS cell lines (143B, U-2 OS, MG-63, MNNG and Saos-2) were purchased from Jennio (Guangzhou, China). Osteoblastic hFOB1.19 cells were cultured in DMEM/F-12 medium (Gibco, USA) with 10% fetal bovine serum (FBS) (BI, Israel), 2.5 mM L-glutamine (Invitrogen, USA) and 0.3 mg/ml geneticin (Gibco, USA). OS cells were maintained in DMEM medium with 1% penicillin/streptomycin (Invitrogen, USA) and 10% FBS in a humidified incubator at 37 °C and 5% CO2.
RNA sequencing
High-throughput sequencing was performed to identify circRNAs according to the following steps. Total cellular RNA was isolated from 3 matched OS tissues and adjacent non-cancerous tissues with the RNAiso Plus reagent kit (TaKaRa, Japan). Ribosomal RNA was eliminated based on the Ribo-Zero Magnetic Kit (Epicentre, USA), whereas linear RNA digestion was performed using RNase R (Epicentre, USA). The RNAs were broken into small pieces using a fragmentation buffer (Ambion, USA) to generate templates for reverse transcription. Then cDNA was purified with magnetic beads and eluted with EB buffer. Fragments of suitable sizes were selected following agarose gel electrophoresis before amplification to generate the cDNA library. Sequencing of the prepared libraries was conducted on the Illumina HiSeq 2500 platform.
Measurement of RNAs expression
For qRT-PCR analysis of circRNAs and mRNAs, cDNA was synthesized by reverse transcription using the PrimeScrip RT Reagent kit (TaKaRa, Japan). Real-time amplification was conducted using SYBR Premix Ex Taq II (TaKaRa, Japan) in the LightCycler 96 System (Roche, Germany). For miRNA analysis, reverse transcription was achieved using stem-loop RT primers specific for miRNA (GeneChem, China) based on the Evo M-MLV RT kit (Accurate Biology, China), followed by qPCR with SYBR Green Premix Pro Taq HS qPCR kit (Accurate Biology, China). U6 (for miRNA) and GAPDH (for circRNA and mRNA) served as the endogenous controls. Each experiment was repeated thrice. Expression results were analyzed based on the 2-ΔΔCt method. Primer sequences are provided in the Additional file 1: Table S1.
RNase R digestion and nucleic acid electrophoresis
Total RNA (2 μg) was extracted and digested with RNase R (3 U/μg) for 15 min at 37 °C. The control group underwent the same processes as the experimental samples except for the addition of RNase R. cDNA and real-time PCR assay were performed as aforementioned. Additionally, circular and linear transcripts were amplified using specific divergent and convergent primers with or without RNase R. The PCR products with cDNA or genomic DNA (gDNA) as templates were analyzed using 2% agarose gel electrophoresis. DNA fragment bands were visualized with UV trans-illumination and images were taken.
Actinomycin D assay
Briefly, OS cells were maintained in six-well plates for 24 hours after which they were treated with new medium supplemented with 2 μg/mL actinomycin D (Sigma-Aldrich, USA). Total cellular RNA for qRT-PCR analysis was extracted at 0, 4, 8, 12 and 24 hours following actinomycin D treatment.
Nuclear-cytoplasmic fractionation
Extraction and purification of cytoplasmic and nuclear RNAs were performed with the PARIS kit (Life Technologies, USA) as per the manufacturer’s protocols. Next, qRT-PCR was performed to quantify the expression of linear and circular RNAs, with U6 and GAPDH serving as internal references for the nuclear and cytoplasmic RNA, respectively.
Fluorescence in situ hybridization (FISH)
RNA-FISH assay was performed to uncover the subcellular localization of circ_001422. Cy3-labeled circ_001422 probe was constructed by RiboBio (Guangzhou, China). The fluorescent signal was generated using the Fluorescent in Situ Hybridization kit (RiboBio, China) and a Nikon laser scanning confocal microscope was utilized to take pictures.
Oligonucleotides, plasmids, cell transfection and lentiviral infection
The miR-195-5p mimics/inhibitors, miR-195-5p agomir/antagomir and their negative controls (NC) were purchased from RiboBio (Guangzhou, China). The short hairpin RNAs (shRNAs) and NC vectors for circ_001422 and FGF2 silencing were constructed by Geneseed (Guangzhou, China). The sequences of the most effective shRNAs (sh-circ_001422#2 and sh-FGF2#1) and their NC vectors were cloned into the recombinant lentiviral vectors and thereafter packaged into viral particles. For overexpression lentiviruses, the full-length cDNA sequences of circ_001422 and FGF2 were PCR amplified and cloned into the lentiviral overexpression vectors constructed by Geneseed (Guangzhou, China). Transient transfections were performed using the EndoFectin Max reagent (Genecopoeia, USA). At 48 h post-lentivirus infection, OS cells were subject to puromycin (2 μg/mL, Invitrogen, USA) or geneticin (500 μg/ml, Gibco, USA) selection for 2 weeks to construct the stable cell lines. Knockdown or overexpression efficiency was verified using qRT-PCR.
Cell proliferation assays
Cell viability of 143B and Saos-2 cells after transfection was assessed using the 5-ethynyl-2’-deoxyuridine (EdU) incorporation assay and colony formation assay. The EdU Apollo 488 kit (RiboBio, China) was utilized to conduct the EdU incorporation assay. Using a Nikon inverted fluorescence microscope, EdU-positive cells were stained green and total nuclei were stained blue.
To evaluate the ability of cells to form colonies, transfected cells were counted and seeded into 6-well plates at the density of 550 cells/well. After 10 days of cultivation, cells were stained using 0.1% crystal violet (Solarbio, China) and the colony numbers were counted.
Flow cytometry
Assessment of the cell cycle of transfected cells was performed by the following procedure. In brief, the cultured cells were harvested, washed twice in phosphate buffer (PBS) and fixed overnight at 4 °C using pre-cooled 75% ethanol. After staining with propidium iodide, cell cycle distribution was determined by a BD flow cytometer. The 4’,6-diamidino-2-phenylindole (DAPI) and Annexin-V-allophycocyanin (APC) double staining kit (BestBio, China) were used for cell apoptotic analyses.
Transwell assays
The migratory and invasive abilities of OS cell lines were evaluated using Transwell migration chambers (Costar, USA) and Transwell invasion chambers pre-coated with 50 μl of 2 mg/ml Matrigel (BD Biosciences, USA), respectively. Briefly, transfected cells (4 × 104 cells/well for migration, 8 × 104 cells/well for invasion) suspended in 200 μl of serum-free DMEM medium were seeded into the upper chambers. Meanwhile, 600 μL of DMEM medium supplemented with 10% FBS used as the attractant was introduced into the lower chambers. After a 24-h culture, cells adhering to the lower surface of the membranes were fixed with paraformaldehyde (4%) and stained using crystal violet (0.1%), whereas cotton swabs were used to wipe off cells on the upper surface. At least three random fields of cells that had migrated or invaded to the lower surface were photographed under an inverted light microscope.
Western blot
Total proteins of homogenized tissues or cell lysates were extracted using ice-cold RIPA solution (Fudebio, China) and protease inhibitors (Fudebio, China). The proteins were diluted to equal concentrations, denatured in boiling water bath, detached by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat milk in Tris-buffered saline-Tween (TBST) buffer and then incubated overnight at 4 °C with several primary antibodies against cleaved CASP3 (1:1000) (Affinity Biosciences, USA), CCND1 (1:1200) (Proteintech, China), CDK4 (1:2000) (Abcam, USA), BAX (1:1000) (Abcam, USA), BCL2 (1:800) (Abcam, USA), E-cadherin (1:2000) (Proteintech, China), N-cadherin (1:2000) (Proteintech, China), Vimentin (1:3000) (Proteintech, China), FGF2 (1:200) (Santa Cruz Biotechnology, USA), PI3K (1:1000) (Cell Signaling Technology, USA), phosphorylated PI3K (p-PI3K, 1:1000) (Cell Signaling Technology, USA), Akt (1:1000) (Cell Signaling Technology, USA), phosphorylated Akt (p-Akt, 1:1000) (Cell Signaling Technology, USA) and GAPDH (1:10000) (Proteintech, China). After four washes with TBST buffer, the membranes were re-incubated for 60 min at 25 °C with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10000) (Bioss, China). The protein bands were visualized with a chemiluminescence imaging system (Bio-Rad, USA).
Animal models
The protocols for animal experiments were approved by the Medical Ethics Committee of Southern Medical University. Four weeks old female BALB/c athymic nude mice were purchased from Guangdong Medical Laboratory Animal Center (Guangdong, China) and reared under pathogen-free facility with adequate standard food and water. A subcutaneous xenograft tumor model was established by subcutaneously injecting 5 × 106 143B stable cells into the right limbs of nude mice (n = 5 per group). The tumor volume was measured every 3 days and calculated using the formula: volume = length × width2 × 0.5. For the lung metastatic model, the abovementioned cells were injected into nude mice via the tail vein (2 × 106 143B cells/ per mouse, n = 5 per group). To evaluate the effect of miR-195-5p in vivo, the miR-195-5p antagomir/agomir or negative controls were injected intratumorally (for the subcutaneous xenograft tumor model) or intravenously via tail vein (for the lung metastatic model) twice a week for 2 weeks, following the manufacturer’s recommendations (RiboBio, China). Tumor tissues were extracted from sacrificed mice four weeks after inoculation.
Hematoxylin and eosin (H&E) staining
Paraffin-embedded thin 4-μm lung sections containing metastatic nodules were dewaxed using xylene and thereafter rehydrated using gradient alcohol. Then the sections were stained using hematoxylin and eosin for general histological examination by standard procedures.
Tissue expression of target proteins
The expression of target proteins in tissue samples of OS patients or xenograft animal models was determined using immunohistochemistry (IHC) as previously described [17]. The tissues were incubated with primary antibodies against FGF2 (1:100), Ki-67 (1:200), PCNA (1:300), N-cadherin (1:200), E-cadherin (1:200), Vimentin (1:300). Apart from FGF2 (Santa Cruz Biotechnology, USA) the rest of the antibodies were purchased from the same manufacturer (Proteintech, China). After washing the tissue sections, they were re-incubated in the presence of HRP-conjugated secondary antibodies (1:200) (Servicebio, China) and thereafter stained with Diaminobenzidine (Zhongshan Golden Bridge, China). The tissue images were observed and captured using an orthophoto microscope.
TUNEL assay
To assess apoptotic DNA fragmentation, xenograft tumor tissues were first fixed for 24 h with 4% paraformaldehyde and thereafter embedded in paraffin. Apoptosis in situ was evaluated based on the TUNEL Apoptosis Assay kit (Alexa Fluor 488) (Yeasen, China). Corresponding photos of the apoptotic cells were captured using a fluorescence microscope. The analyses were performed in at least three random fields for each sample.
Bioinformatics analysis
To predict the potential miRNAs binding with circ_001422, five online bioinformatics tools were used; starBase (http://starbase.sysu.edu.cn/), miRanda (http://www.microrna.org/), TargetScan (http://www.targetscan.org/vert_72/), RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/), and RNA22 (https://cm.jefferson.edu/rna22/). The potential downstream target mRNAs for miR-195-5p were predicted following analysis of miRanda, RNAhybrid, TargetScan and miRTarBase (http://mirtarbase.mbc.nctu.edu.tw/) databases.
RNA immunoprecipitation (RIP)
Transfected cells were washed twice in ice-cold PBS and thereafter lysed in RIP lysis solution containing RNase and protease inhibitors. Incubation of the cell lysates (200 μl) was done overnight at 4 °C with immunoprecipitation buffer containing anti-Argonaute2 (anti-Ago2) conjugated magnetic beads (Millipore, USA) or negative anti-IgG (Millipore, USA). Subsequently, the immunoprecipitated RNAs were extracted and purified to determine the abundance of target RNAs using qRT-PCR.
RNA pull-down assay
A control probe and a biotinylated circ_001422 probe were constructed by RiboBio (Guangzhou, China). The probes were coated with C-1 magnetic beads (Life Technologies, USA) following 2 h incubation at room temperature with the aforementioned beads. The transfected cells were harvested and treated with ice-cold lysis solution, and thereafter incubated overnight at 4 °C with circ_001422 or oligo probes. Finally, the pull-down products were extracted and purified using the RNeasy Mini kit (Qiagen, USA). The abundance of circ_001422 and miRNAs in the RNA complexes was evaluated by qRT-PCR.
Luciferase reporter assay
The circ_001422 or FGF2 fragments with the mutant-type (MUT) or wild-type (WT) miR-195-5p binding sites were subcloned downstream of the Renilla gene in psiCHECK-2 dual-luciferase reporter vector (Geneseed, China). The 143B and Saos-2 cells in the logarithmic growth phase were co-transfected with reporter vectors and miR-195-5p mimics or NC mimics. After 48 h incubation, the Dual-Luciferase Reporter Assay System (Promega, USA) was utilized to measure the luciferase activity.
Statistical analysis
Continuous variables were presented as mean ± standard deviation. All analyses were performed using SPSS 20.0 software (IBM, USA). Difference between groups was analyzed using unpaired Student’s t-test, or one-way analysis of variance (ANOVA) and Tukey’s test. Survival was analyzed by Kaplan-Meier curves and log-rank test. The correlations among circ_001422, miR-195-5p and FGF2 were determined using Pearsons correlation analyses. P < 0.05 was considered significant.