The study included 60 OS patients (37 males and 23 females; 12 to 33 years; 23.2 ± 3.4 years) who admitted to The First People's Hospital of Lianyungang between July 2016 and July 2019. The inclusion criteria were (1) OS patients diagnosed for the first time and (2) no therapy was initiated. The exclusion criteria were (1) patients complicated with other severe diseases, (2) patients with blood relationship and (3) recurrent cases. During biopsy, OS tissues and paired adjacent noncancerous tissues (normal bone tissues within 5 cm around tumors) were collected from each patient and freshly stored in liquid nitrogen before use. All tissue samples were confirmed by histopathological biopsy. The Ethics Committee of the aforementioned hospital approved this study (Ethical Approval No. #8631). Informed consent was obtained. Characteristics of patients are shown in Additional file 1: Table S1.

Human OS cell lines U2OS, MG-63, HOS, SJSA-1, and 143B (ATCC, USA) and normal osteoblast cells hFOB1.19 (ATCC, USA) were included in this study. MG-63, HOS, hFOB1.19 and 143B cell lines and SJSA-1 cells were cultured in EMEM (ATCC, USA) with 10% FBS and RPMI-1640 medium (ATCC, USA) with 10% FBS, respectively, at 37 °C (or at 33.5 °C for hFOB1.19 cells) in an incubator with 5% CO2 and 95% humidity.

PCED1B-AS1 siRNA (5′-AAGCGGUUCUCGUGCCUCAGU-3′), NC siRNA (Cat# SIC001, Sigma-Aldrich), miR-10a (5′-UACCCUGUAGAUCCGAAUUUGUG-3′) or NC miRNA (5′-GGUUCGUACGUACACUGUUCA-3′) was transfected into cells using Lipofectamine® 2000 (Invitrogen). In each transfection, 1 × 107 cells in a 10-cm dish were transfected with 50 nM miRNA and/or siRNA. NC siRNA- or NC miRNA-transfected cells were NC cells. Untransfected cells were control (C) cells. Subsequent experiments were performed at 48 h of post-transfection.

Total DNAs were extracted from U2OS and MG-63 cells using Quick-DNA Kit (ZYMO RESEARCH) and converted. The converted DNA samples were used as templates to perform MSP using 2X Taq FroggaMix (FroggaBio, USA) to analyze the methylation status of miR-10a gene with primers 5′-TTATTATTGTGTGTTCGGAAAATC-3′ (forward) and 5′-GTAACGCGCCTAACTATTTAACA-3′ (reverse) for methylated DNAs and 5′-TGTTTATTATTGTGTGTTTGGAAAATT-3′ (forward) and 5′-TCATAACACACCTAACTATTTAACAA-3′ (reverse) for un-methylated DNAs. PCR product was 1601 bp (from position -101 to -1701). PCR conditions were 5 min at 95 °C followed by 35 cycles of 95 °C for 30 s, 55 °C for 28 s and 72 °C for 35 s and a final extension at 72 °C for 10 min. All PCRs were conducted on a Bio-Rad C1000 PCR machine (Bio-Rad).

After RNA preparation using Direct-zol RNA, DNase I-digested RNA samples were used to prepare cDNA samples. With cDNA samples as templates, qPCRs were performed with 18S rRNA internal control to measure the accumulation levels of both PCED1B-AS1 and miR-10a. The method of 2−∆∆Ct was used for data normalizations. The following specific primers were employed: PCED1B-AS1 forward 5′-AAGGGGAAAGGAGGAAGTGAGAAG-3′ and reverse 5′-GGAAGCCAGTGAGCCAGGAGT-3′ and miR-10a forward 5′-CAGTGCAGGGTCCGAGGT-3′ and reverse 5′-GCCGTAC CCTGTAGATCCGAA-3′. PCR conditions were 1 min at 95 °C followed by 40 cycles of 95 °C for 10 s and 57 °C for 40 s. All qPCRs were conducted on BioRad CFX96 Touch Real Time PCR (Bio-Rad).

The proliferation of both U2OS and MG-63 cells after transfections was analyzed using a CCK-8 kit (Dojindo). Briefly, cells were harvested and cultured at 37 °C in a 96-well plate with 3000 cells in 0.1 ml medium per well. Three replicate wells were set for each experiment. Cell culture was performed for 48 h, followed by adding CCK-8 solution to 10%. After incubation with CCK-8 for 4 h, OD values at 450 nm were measured.

Transwell Inserts (8.0 μm, Corning) were used and cells in

non-serum medium were added to the upper chamber. To induce cell movement, FBS was added to 20% in the lower chamber, and the upper chamber was filled with 6000 cells in serum-free media. After incubation at 37 °C for 24 h, cells on the lower membranes were stained with 1% crystal violet (Sigma-Aldrich) and counted.

Three independent replicates were included in each experiment, and mean ± SD values were used to express the data. Paired tissues (paired t test) and multiple groups (ANOVA Tukey’s test) were compared. The 60 OS patients were divided into high and low PCED1B-AS1 level groups (n = 30, cutoff value = median level of PCED1B-AS1 in OS tissues). Chi-squared test was applied to explore the associations between patients’ clinical characteristics and PCED1B-AS1 expression levels. Correlations were explored by performing Pearson’ correlation coefficient. p < 0.05 was statistically significant.