The expression levels of KIAA1429 and ZFPM2-AS1 in sarcomas and normal tissue were analyzed using data from The Cancer Genome Atlas (TCGA) via the UALCAN tool (https://ualcan.path.uab.edu/index.html) (24). The association between the expression of ZFPM2-AS1 in sarcoma tissues and the overall survival of patients was analyzed using TCGA data via the Gene Expression Profiling Interactive Analysis 2 (GEPIA2) database (http://gepia2.cancer-pku.cn/#index) (25). The expression of KIAA1429 in 18 pairs of osteosarcoma and non-tumoral tissues was analyzed using the GSE99671 osteosarcoma-related dataset from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/info/geo2r.html). The correlation between the expression of KIAA1429 and other genes in 87 osteosarcoma tissue samples from TCGA-TARGET-osteosarcoma (OS) database was analyzed using PDX for Childhood Cancer Therapeutics online analysis software. The 20 genes identified as having the highest expression correlation were subsequently downloaded and collated. The correlation between the expression of KIAA1429 (probe ID: 25962) and ZFPM2-AS1 (probe ID: 102723356) was analyzed in 52 samples from the GEO osteosarcoma-related dataset GSE87624. Sarcoma data were also downloaded from TCGA database to analyze the correlation between KIAA1429 and ZFPM2-AS1 expression in 260 sarcoma tissue samples. The gene sequence of ZFPM2-AS1 (NR_125796.1) was retrieved from the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/gene/?term=ZFPM2-AS1). Finally, m⁶A modification sites in the ZFPM2-AS1 sequence were predicted using SRAMP (http://www.cuilab.cn/sramp/) online bioinformatics software (26).

A total of 20 pairs of surgically resected osteosarcoma and paraneoplastic tissue samples were collected from the Central Hospital Affiliated to Shenyang Medical College (Shenyang, China) and Liaoning Provincial Cancer Hospital (Shenyang, China). The samples were collected from 8 male and 12 female patients (median age, 16 years; age range, 8–22 years) who underwent surgery between April 2016 and April 2022. All patients were clinically and pathologically diagnosed with osteosarcoma. Written informed consent was obtained from all adult participants and from the parents or legal guardians of participants <18 years of age. The study protocol was approved by the Medical Ethics Committee of the Central Hospital Affiliated to Shenyang Medical College (approval no. 2022DEC12-7).

The hFOB1.19 human osteoblast cell line and the 143B and MG63 osteosarcoma cell lines were obtained from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. Characterization of the three cell lines using short tandem repeat analysis demonstrated that these cells were not cross-contaminated. The hFOB1.19 osteoblasts were cultured in Gibco^® DMEM/F12 medium, the MG63 cells were cultured in Minimal Essential Medium, and the 143B cells were cultured in RPMI-1640 medium (all from Thermo Fisher Scientific, Inc.). The media were supplemented with 10% (v/v) Gibco fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and a solution of penicillin and streptomycin (penicillin, 100 U/ml; streptomycin, 0.1 mg/ml). All three cell lines were cultured in a cell culture incubator in an atmosphere containing 5% CO₂. The hFOB1.19 cells were maintained at 34°C, while the 143B and MG63 cells were cultured at 37°C.

Total RNA was extracted from cells and tissues using an Invitrogen TRIzol^® Plus RNA Purification Kit (Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. Reverse transcription was performed with the PrimeScript RT Master Mix (Takara Bio, Inc., cat. no. RR036A) in a 20 µl reaction containing 1 µg total RNA, 5X RT Master Mix, and RNase-free water. The thermal protocol included 37°C for 15 min, 85°C for 5 sec, and a final hold at 4°C. qPCR was carried out using the TB Green Premix Ex Taq II kit (cat. no. RR820A; Takara Bio, Inc.) under the following conditions: Initial denaturation at 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. GAPDH served as the internal reference gene, and relative gene expression was calculated using the 2^−ΔΔCq method. All primers were synthesized by Takara Bio, Inc., and sequences are listed in Table SI.

Proteins were extracted from cells and tissues using a protein extraction kit (cat. no. GM1001; Wuhan Servicebio Technology Co., Ltd.). Protein concentrations were determined using a BCA protein assay kit (Wuhan Servicebio Technology Co., Ltd.). A total of 25 µg protein per lane was denatured in a water bath at 100°C for 5 min, followed by separation using 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; cat. no. G2176; Wuhan Servicebio Technology Co., Ltd.) at a constant voltage of 120 V. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes (cat. no. G6044-0.45; Wuhan Servicebio Technology Co., Ltd.) at a constant current of 400 mA. Membranes were blocked with NcmBlot Rapid Blocking Buffer (cat. no. P30500; NCM Biotech) at room temperature (25°C) for 15 min. Primary antibodies against KIAA1429 (cat. no. ab271136; Abcam; 1:1,000 dilution) and GAPDH (cat. no. ab9485; Abcam; 1:2,500 dilution) were incubated with the membranes at 4°C overnight. After washing with Tris-buffered saline containing 0.1% Tween 20 (TBST), membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (cat. no. ab205718; Abcam; 1:10,000 dilution) at room temperature for 1 h. Protein bands were visualized using an ultra-sensitive ECL chemiluminescence detection kit (cat. no. G2074; Wuhan Servicebio Technology Co., Ltd.) and imaged with a gel documentation system (ChemiScope 6100; Shanghai Qinxiang Scientific Instrument Co., Ltd.). Protein expression levels were quantified using ImageJ software (Version 1.53; National Institutes of Health) and normalized to GAPDH.

Two short hairpin RNAs (shRNAs) targeting the KIAA1429-specific junction region (shKIAA1429-1 and shKIAA1429-2), their corresponding negative control shRNAs (shNCs), a KIAA1429 overexpression plasmid (oeKIAA1429), and the empty plasmid vector pCMV6-Entry were designed and synthesized by Hanheng Biotechnology (Shanghai) Co., Ltd. shRNAs (final concentration: 50 nM) and plasmids (final concentration: 2 µg/ml) were transfected into 143B, MG63, and hFOB1.9 cells using the Invitrogen Lipofectamine^® 3000 reagent (cat. no. L3000001; Thermo Fisher Scientific, Inc.) under standard conditions (37°C for 6 h), followed by replacement with complete culture medium. Additionally, a small interfering RNA (siRNA) targeting ZFPM2-AS1 (siZFPM2-AS1) and its negative control siRNA (siNC) were designed by Guangzhou RiboBio Co., Ltd. and transiently transfected into osteosarcoma cells at a final concentration of 50 nM using the RiboFect^® CP Transfection Kit (cat. no. C10511-05; Guangzhou RiboBio Co., Ltd.), according to the manufacturer's protocol. Cells were harvested 48 h post-transfection for subsequent experiments. The sequences of shRNAs, siRNAs, and oeKIAA1429 are listed in Table SI.

143B and MG63 cells were inoculated into 96-well plates at a density of 2×10³ cells/well, and cultured in a cell culture incubator at 37°C in an atmosphere containing 5% CO₂. At 24, 48, 72 and 96 h, 10 µl CCK-8 solution (cat. no. G4103; Wuhan Servicebio Technology Co., Ltd.) was added to each well for 2 h. Following this, the 96-well plates were removed from the incubator and the absorbance at 450 nm was measured using a microplate reader (Multiskan FC Photometer; Thermo Fisher Scientific, Inc.).

Different densities of the 143B and MG63 cells were used, according to whether the Transwell assay was used to assess migration (4×10⁴ cells) or invasion (8×10⁴ cells). The cells were cultured in the upper chamber of the Transwell apparatus (Corning, Inc.). After 24 h at 37°C, the cells in the upper chamber were scraped off and those on the lower side of the Transwell membrane were fixed with anhydrous ethanol prior to staining with 0.1% crystal violet staining solution (cat. no. G1014; Wuhan Servicebio Technology Co., Ltd.) for 30 min at room temperature. Following a rinse with running water, the migrated or invaded cells were observed under an inverted microscope (Olympus Corporation). For invasion assays, the upper chambers of Transwell inserts (Corning, Inc.) were pre-coated with Matrigel Basement Membrane Matrix (Corning, cat. no. 354234) diluted 1:8 in serum-free medium, followed by polymerization at 37°C for 1 h. Cell densities were adjusted according to assay type: 4×10⁴ cells/well for migration and 8×10⁴ cells/well for invasion. Cells were suspended in serum-free RPMI-1640 medium (for 143B) or MEM (for MG63) and added to the upper chamber. The lower chamber contained complete medium with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.) as a chemoattractant. After 24 h incubation at 37°C in 5% CO₂, non-invaded cells on the upper membrane surface were removed using a cotton swab. Cells on the lower side were fixed with anhydrous ethanol for 15 min, stained with 0.1% crystal violet (Wuhan Servicebio, cat. no. G1014) for 30 min, and rinsed with PBS. Images were captured using an inverted microscope (Olympus Corporation) at 200× magnification. Cell counts were quantified from five random fields per membrane.

The assay was conducted using an m⁶A-Methylated RNA Immunoprecipitation Kit (cat. no. 11096.6; Guangzhou Ribobio Co., Ltd.) in accordance with the manufacturer's instructions. In brief, the collected RNA was fragmented using RNA Fragmentation Buffer. A fraction (one-tenth) of the fragmented RNA was retained as an input control group for subsequent analysis, while the remainder was used for m⁶A immunoprecipitation (IP). Anti-m⁶A magnetic beads were prepared using m⁶A antibody (cat. no. ab151230; dilution, 1:500). The fragmented RNA was added to the MeRIP reaction mixture, and incubated with anti-m⁶A magnetic beads. After incubation, the mixture was centrifuged at 1,000 × g for 3 min at 4°C and the supernatant was discarded. The magnetic beads were then washed, followed by elution of the bound RNA. The eluted RNA fragments were recovered and purified using the GeneJET RNA Purification Kit (cat. no. K0731; Thermo Fisher Scientific, Inc.). The recovered RNA was designated as the IP group. Subsequently, the input and IP groups were analyzed by RT-qPCR.

The collected cells (2×10⁶ cells) were transferred to 6-well plates and cultured to a confluency of 80%. The cells were then treated with 2 µg/ml actinomycin D reagent (cat. no. 50-76-0; MedChemExpress). Total cellular RNA was extracted at 0, 4, 8 and 12 h and analyzed by RT-qPCR.

Statistical analyses were performed using GraphPad Prism 8.0 software (Dotmatics). Results are expressed as the mean ± standard deviation. For comparative analyses involving two groups, paired tests were used to evaluate results from paired patient tissue samples: a paired t-test was applied for normally distributed data, whereas the Wilcoxon signed-rank test was applied for non-normally distributed data. Unpaired t-tests were used for the comparison of two independent samples with normally distributed means. In datasets comprising three or more groups, one-way analysis of variance followed by Tukey's post hoc test was used for normally distributed data, and Kruskal-Wallis test was performed followed by Dunn's post hoc analysis for non-normally distributed data. Linear correlation analyses were performed, with Pearson correlation coefficients calculated for normally distributed data and Spearman correlation coefficients for non-normally distributed data. Each experiment was replicated thrice. P<0.05 was considered to indicate a statistically significant difference.

Bioinformatics analysis was performed to investigate the expression of KIAA1429 in osteosarcoma and normal tissues. Analysis of TCGA data revealed that the expression of KIAA1429 in sarcoma was markedly elevated compared with that in normal tissues. The expression of KIAA1429 was also analyzed in the GSE99671 dataset from the GEO database, which revealed that the expression of KIAA1429 in sarcoma tissues was significantly higher compared with that in the corresponding normal tissues (Fig. 1A and B). Subsequently, 20 pairs of osteosarcoma and paraneoplastic tissues collected from patients were examined by RT-qPCR analysis, which revealed a significant upregulation of KIAA1429 expression in the osteosarcoma tissues (Fig. 1C). In addition, the western blot analysis of five pairs of osteosarcoma and paraneoplastic tissues confirmed that expression of KIAA1429 protein was significantly upregulated in the osteosarcoma tissues (Fig. 1D). Subsequently, normal osteoblast and osteosarcoma cell lines were analyzed using RT-qPCR and western blotting. The results demonstrated that KIAA1429 mRNA and protein expression levels in the 143B and MG63 osteosarcoma cells were significantly higher compared with those in the hFOB1.19 cells (Fig. 1E and F). The findings of the bioinformatics analysis, tissue and cellular experiments collectively demonstrate that KIAA1429 expression is upregulated in osteosarcoma.

The impact of KIAA1429 on osteosarcoma cell proliferation, migration and invasion was investigated in functional assays of 143B and MG63 cells transfected with shKIAA1429-1, shKIAA1429-2 and shNC. The successful establishment of KIAA1429 knockdown was verified in the two cell lines via RT-qPCR and western blotting (Fig. 2A-D). A CCK-8 cell proliferation assay was then performed to assess the impact of KIAA1429 knockdown on cell proliferation. The results demonstrated that KIAA1429 knockdown significantly inhibited the proliferative capability of the 143B and MG63 cells compared with that of the corresponding shNC controls (Fig. 2E and F). Subsequently, Transwell assays were performed to evaluate the migration and invasion of the 143B and MG63 cells. The results revealed that the migratory and the invasive capabilities of the cells were significantly diminished following the knockdown of KIAA1429 (Fig. 2G and H). Together, these findings suggest that KIAA1429 promotes the proliferation, migration and invasion of 143B and MG63 osteosarcoma cells.

Expression data from 87 osteosarcoma tissues in TCGA-TARGET-OS database were analyzed to identify genes whose expression levels significantly correlated with KIAA1429 expression levels. Of the top 20 genes whose expression correlated with that of KIAA1429, ZFPM2-AS1 was the only lncRNA identified (Table SII and Fig. 3A). The online tool SRAMP was then used to predict the presence of theoretical m⁶A modification sites (RRACH sequences) in the ZFPM2-AS1 sequence, and six such sites were identified (Fig. S1A). Based on this finding, the potential regulation of ZFPM2-AS1 by KIAA1429 was investigated. ZFPM2-AS1 expression levels were found to correlate with KIAA1429 expression levels in the GSE87624 osteosarcoma-related dataset as well as in sarcoma tissues in TCGA database (Fig. 3B and C). Similarly, reduced expression levels of ZFPM2-AS1 were observed following KIAA1429 knockdown in 143B and MG63 cells (Fig. 3D and E). hFOB1.19 cells overexpressing KIAA1429 were successfully constructed, as confirmed by western blotting (Fig. S1B). RT-qPCR results revealed a corresponding increase in the expression level of ZFPM2-AS1, further supporting a positive association between KIAA1429 and ZFPM2-AS1 expression (Fig. S1C). Although ZFPM2-AS1 has been characterized as an oncogene in numerous types of tumor, its specific role in osteosarcoma remains unclear. Analysis of data from TCGA revealed that ZFPM2-AS1 expression is upregulated in sarcoma tissue compared with normal tissue, and high ZFPM2-AS1 expression is associated with a poorer overall survival profile in patients with sarcoma (Fig. 3F and G). Subsequently, the expression levels of ZFPM2-AS1 were assessed in 20 pairs of osteosarcoma and paraneoplastic tissues via RT-qPCR analysis. The results demonstrated that the expression of ZFPM2-AS1 in osteosarcoma tissues was upregulated compared with that in paraneoplastic tissues (Fig. 3H). Furthermore, analysis of the expression of ZFPM2-AS1 in hFOB1.19, 143B and MG63 cells. revealed that ZFPM2-AS1 expression in the osteosarcoma cell lines was elevated compared with that in the normal osteoblast cell line (Fig. 3I). Taken together, these results provide evidence to support a correlation between the expression levels of ZFPM2-AS1 and KIAA1429, and demonstrate that the expression of ZFPM2-AS1 is upregulated in osteosarcoma tissues and cells.

SRAMP predicted m⁶A modification sites at positions 579, 613, 624, 750, 780 and 833 of the ZFPM2-AS1 sequence, spanning ~260 base pairs. (Fig. S1A). Primers were designed based on the gene regions containing these predicted modification sites, and the m⁶A methylation level of ZMIZ1-AS1 in osteosarcoma tissues and cells was determined by MeRIP-qPCR. The results revealed that the m⁶A methylation level of ZFPM2-AS1 was significantly elevated in osteosarcoma tissues and cells in comparison with the levels observed in paraneoplastic tissues and hFOB1.19 cells, respectively (Fig. 3J and K). To further investigate the regulatory role of KIAA1429, the m⁶A methylation levels of ZFPM2-AS1 were examined via MeRIP-qPCR assay in 143B and MG63 cells following KIAA1429 knockdown. The knockdown of KIAA1429 was observed to result in a significant reduction in the m⁶A methylation level of ZFPM2-AS1 compared with that in the shNC group (Fig. 3L and M). In addition, an actinomycin D assay revealed that the knockdown of KIAA1429 shortened the half-life of ZFPM2-AS1, indicating that its stability was reduced (Fig. 3N and O). Considered together, these findings suggest that m⁶A methylation levels of ZFPM2-AS1 are elevated in osteosarcoma tissues and cells. Furthermore, based on the observed reduction following KIAA1429 knockdown, the results suggest that KIAA1429 may increase the m⁶A methylation levels of ZFPM2-AS1 and promote its stability.

Finally, the impact of ZFPM2-AS1 on KIAA1429-mediated osteosarcoma proliferation, migration and invasion was investigated. First, a cell model with stable overexpression of KIAA1429 was established in 143B and MG63 cells, and the successful construction of the model was verified via western blotting (Fig. 4A and B). Subsequently, siZFPM2-AS1 was transfected into 143B and MG63 cells, resulting in significantly reduced ZFPM2-AS1 expression levels compared with those in the respective siNC-transfected cells, as validated by RT-qPCR (Fig. S1D and E). Then, siZFPM2-AS1 was transfected into 143B and MG63 cells overexpressing KIAA1429, and ZFPM2-AS1 expression was quantified by RT-qPCR. The expression level of ZFPM2-AS1 was observed to increase following the overexpression of KIAA1429 compared with that in the empty vector control group, which was consistent with the observed reduction in ZFPM2-AS1 expression following KIAA1429 knockdown (Fig. 4C and D). However, this oeKIAA1429-induced increase in ZFPM2-AS1 expression was attenuated by co-transfection with siZFPM2-AS1 (Fig. 4C and D). Subsequently, CCK-8 and Transwell functional assays were performed. The overexpression of KIAA1429 significantly promoted cell proliferation, migration and invasion compared with that of the vector control cells. However, these effects of KIAA1429 were attenuated following the knockdown of ZFPM2-AS1 in KIAA1429 overexpressing cells (Fig. 4E and H). These findings suggest that the knockdown of ZFPM2-AS1 attenuates the effects of KIAA1429 on the proliferation, migration and invasion of 143B and MG63 cells.