Human tissue samples
We collected 31 pairs of specimens consisting of osteosarcoma and adjacent normal tissues from osteosarcoma patients (from January 2013 to November 2019) at The Tumor Hospital of Harbin Medical University. The Ethics Committee of The Affiliated Hospital of Harbin Medical University approved this study, and we obtained written informed permission from all the patients. Clinicopathological parameters of OS patients were showed in Table1. All tissues were harvested and then kept at -80℃ or snap-frozen in liquid nitrogen immediately pending use.
Cell culture
We bought the normal human osteoblast cell line hFOB1.19 and osteosarcoma cell lines (143B, U2OS, MG63, and HOS) from the American Type Culture Collection (Manassas, VA). We cultured all the cell lines in Dulbecco’s modified Eagle’s medium (DMEM, (HyClone, Logan, USA) added with 10% fetal bovine serum (FBS, Gibco, NY, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Baomanbio, Shanghai, China) and incubated in an incubator (5% CO2, 37°C).
Cell transfection
The FEZF1-AS1-siRNA, miR-4456 mimics, miR-4456 inhibitor, GALNT10 plasmids, and their corresponding negative controls (shNC, NC mimics, NC inhibitor, pcDNA3.1) were constructed and provided by RIBIOBIO (Guangzhou, China). We grew the OS cells in the medium until a confluence of approximately 60-70%. After that, we transfected all the plasmids mentioned above into 143B or U2OS using the riboFECT CP Transfection Kit (C10511-1, RIBIOBIO, Guangzhou, China) as per instructions of the manufacturer. After 48h or 72 h following transfection, we attained the OS cells for downstream experiments.
RT-qPCR
To assess the expression of FEZF1-AS1, GLANT10, and miR-4456 in OS tissue or cells, we performed RT-qPCR. The trizol reagent (Cat: 15596026, Invitrogen, Carlsbad, USA) was utilized to extract total RNA as per the protocol of the manufacturer. The TransScript Green One-Step qRT-PCR SuperMix kit ( Takara, Kyoto, Japan) was used to assay for the relative gene expression via qPCR. GAPDH or U6 was used as an internal control. The 2−ΔΔ‐Ct approach was used to examine the relative level of expression of genes in OS. The sequences of the primers used in this study are shown in Table S1.
Western blot assay
We harvested the treated cells and lysed the using RIPA (Radio Immunoprecipitation Assay, Beyotime, Shanghai, China) buffer. We separated the cell lysates on 12.5% SDS/PAGE (sodium dodecyl sulfate-polyacrylamide), then transferred onto PVDF (polyvinylidene fluoride membrane, Millipore, Billerica, USA) membranes, followed by blocking with 5% non-fat dried milk for 1h at room temperature (RT). After that, we incubated the membranes with primary antibodies at 4℃ against GALNT10, E-cadherin, Vimentin, and GAPDH. Subsequently, we incubated the membranes with the second antibodies separately at RT for 2h, and the proteins detected using an EasyBlot ECL kit (ECL kit, Santa Cruz Biotechnology, Santa Cruz, USA). The antibodies targeting GALNT10, E-cadherin, Vimentin, and GAPDH were all purchased from Abcam Co., Ltd. (Pudong, Shanghai, China). The density of the bands was imaged via enhanced chemiluminescence. GAPDH was employed as an endogenous control.
Bioinformatics Analysis and Luciferase Reporter Assay
We predicted the putative binding sites of miR-4456 and FEZF1-AS1 or GALNT10 via bioinformatics analysis using starbase 2.0 (starbase.sysu.edu.cn). We generated the wild‐type vectors (FEZF1-AS1‐WT and GALNT10‐WT) and mutant‐type vectors (FEZF1-AS1‐Mut and GALNT10‐Mut) using the pGL3 vector (Promega, Madison, WI, USA). In the luciferase reporter test, we used the riboFECT CP Transfection Kit (Catalogue number, manufacturer, country) to co-transfect the U2OS cells with WT or Mut luciferase reporter vectors and miR-4456 mimic or miR-NC mimic. Following 48h, post-transfection, we conducted the luciferase enzyme activity test in each group using the luciferase reporter assay kit (Catalogue number, Promega, country). The luciferase activity of Renilla was employed as the standard.
RNA Immunoprecipitation (RIP)
We harvested 1×107 U2OS and 143B cells with or without miR-4456 mimic and then lysed them in RIP buffer. We performed the RIP test using a Magna RNA immunoprecipitation kit (Millipore, Billerica, MA, USA), as well as magnetic beads, pre-coated with Ago2 or IgG antibody. The levels of FEZF1-AS1 in the complex were assayed via qRT-PCR after the isolation of RNA using the Trizol reagent.
Trans-well assay
Trans-well assays were used to detected OS cell invasion. We used the Trans-well chambers (Corning, NY, USA) to observe OS cell infiltration. We seeded 200µl of OS cell suspension (1 × 105 cells) in the serum-free medium into the upper chamber, which was coated with Matrigel (BD Biosciences, San Diego, CA, USA). After that, we added 800µl of DMEM added with 10% FBS to the lower chamber. Following 48h of incubation, we removed the cells on the top side of the membrane via a swab. We fixed the infiltrative cells on the bottom and stained with 0.1% crystal violet for 15min. The number of invading cells was estimated using a phase-contrast microscope (Olympus, Tokyo, Japan).
Wound healing assays
We seeded the OS cells into 6-well plates and then utilized a sterile 10μl plastic micropipette tip to scratch through the monolayer. After that, we washed the cells thrice using physiological saline, then replaced it with DMEM medium added 0.2% FBS. The cells were scratches for 24h and 48h, then observed the migration of the cells using a microscope (indicate which type of microscope and the manufacturer).
ceRNA modulatory network design and evaluation
The determined prognosis‑correlated lncRNAs and miRNAs were utilized to identify lncRNA‑miRNA modulatory relationships from the correlations using miRcode (mircode.org) and starBase (starbase.sysu.edu.cn/index). Moreover, we selected the miRNA‑mRNA modulatory correlations based on the modulatory information in TargetScan (targetscan.org), miRTarBase (mirtarbase.mbc.nctu.edu.tw) (30), miRanda (microrna.org/microrna), and miRBase (mirbase.org).
Immunohistochemistry
We fixed the tissue samples in 10% formalin and then sliced them into 4μm sections. Subsequently, we incubated the sections at 60ºC for 1h, followed by conventional xylene dewaxing and gradient alcohol dehydration. Then, we blocked the sections blocked using normal goat serum solution at 37°C for 10min, followed by incubation with the primary antibody, anti-GALNT10 for 12h at 4°C. After that, we incubated the sections with the secondary antibody, biotinylated anti-mouse for 10min at RT. The image analysis software (Nikon, Tokyo, Japan) was utilized to count the number of positive cells. The percentage proportion of the positive cells in each field was computed; the percentage of <10% was regarded as negative, and > 10% was considered as positive.
Statistical analysis
All statistical analyses were conducted independently three to six times in the SPSS 20.0 software (Chicago, IL, USA). Student's t-test and ANOVA were applied in determining the significance of differences. A P < .05 signified a marked difference.