In the present study, primary fibroblastic or myofibroblastic tumors from 22 patients that underwent surgical resection at Xiangya Hospital of Central South University (Changsha, China) from 01/01/2017-10/31/2022 were sampled. Patients were aged between 8 and 84 years, and there were 15 male patients and 7 female patients. After surgical resection, all specimens were immediately placed in tissue-protective solution (Biosharp), frozen in liquid nitrogen and stored at −80°C. Tissue samples were used to prepare sections for immunohistochemistry (IHC). Fibrosarcoma tissues from 6 of the 22 cases were also used for mRNA and protein extraction. Clinicopathological characteristics of enrolled patients were obtained from medical records. Tumors were classified histologically using guidelines published in the American Joint Committee on Cancer Limb/Torso Soft Tissue Sarcoma Staging System (8th edition, 2017) (24). The present study was approved by the Ethics Committee of Xiangya Hospital and written informed consent was obtained from all participants or their guardians (ethics approval no. 201603079).

Fibroblastic or myofibroblastic tumor datasets (GSE24369 and GSE21124, respectively) were selected from the Gene Expression Omnibus (GEO) database for gene selection (control group, n=6; tumor group, n=6; from different patients) and GSE21124 (control group, n=9; tumor group, n=34; from different patients).

Human fibrosarcoma HT-1080 cells and human skin fibroblasts (HSF; HSA-S4) were purchased from Abiowell (Changsha Abiowell Biotechnology Co., Ltd.) with STR certification reports. Cells were cultured in high-glucose DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Shanghai ExCell Biology, Inc.) and 1% penicillin/streptomycin at 37°C with 5% CO₂.

IHC was performed as previously described (25). Staining intensity and percentage of positivity were analyzed by ImageJ/Fiji (2.15.0; Github; http://imagej.net/software/fiji/) (26). Staining was evaluated and graded by two independent investigators. The staining intensity scoring was 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). The degree of staining was stratified as 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%) and 4 (76–100%), and was defined as the percentage of positive staining area in the total tumor invasion area. The final score of GALNT12 expression was determined by adding the intensity of staining to the degree of staining; its value was 0–7. The samples were divided into two groups: Low GALNT12 expression (0–3 points) and high GALNT12 expression (4–7 points). Sections were classified into high and low expression groups. The primary antibody used was: Anti-GALNT12 (1:100; cat. no. K108365P; Beijing Solarbio Science & Technology Co., Ltd.). A HRP-conjugated antibody (1:200; cat. no. GB23303; Wuhan Servicebio Technology Co., Ltd. 1:200 for IHC) was used as a secondary antibody.

Immunocytochemistry was performed as previously described (25). The primary antibody used was: Anti-GALNT12 (1:50; cat. no. K108365P; Beijing Solarbio Science & Technology Co., Ltd.). CoraLite594-conjugated Goat Anti-Rabbit IgG (H+L) (1:200; cat. no. SA00013-4; Proteintech Group, Inc.) was used as a secondary antibody.

Total RNA of fibrosarcoma tissues or HT-1080 cells was extracted using TransZol Up (TransGen Biotech Co., Ltd.) according to the manufacturer's instructions. The quality and concentration of the isolated RNA were analyzed using a NanoDdrop 2000 (Thermo Fisher Scientific, Inc.) and reverse transcribed into cDNA using the Evo M-MLV RT kit (Accurate Biology) according to the manufacturer's instructions. RT-qPCR was performed on the Vii7 system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using ribosomal protein S18 (RPS18) as an internal normalization control. Relative mRNA expression was calculated using the 2^−ΔΔCq method (27). Primer sequences are listed in Table SI. For qPCR, 2X SYBR Green qPCR Master Mix (Low ROX) (cat. no. G3321-15; Wuhan Servicebio Technology Co., Ltd.) was used. The recommended qPCR procedure was as follows: Stage 1, 95°C for 30 sec for one cycle; stage 2, 95°C for 15 sec and 60°C for 30 sec for 40 cycles; stage 3, 95°C for 15 sec, 60°C for 60 sec and 95°C for 15 sec for one cycle.

HSF cells, HT-1080 cells and fibrosarcoma tissues were lysed in RIPA buffer (Beyotime Institute of Biotechnology) with protease inhibitors (Wuhan Servicebio Technology Co., Ltd.) (RIPA buffer:protease inhibitors, 100:1) for 15 min on ice to extract total proteins. The BCA method was used to determinate protein concentrations. Subsequently, proteins were boiled with 5X loading buffer (Wuhan Servicebio Technology Co., Ltd.) for 10 min. Nuclear proteins were isolated using a commercial kit (cat. no. R0050; Beijing Solarbio Science & Technology Co., Ltd.) following the manufacturer's protocol. Proteins (50 µg/lane) were separated using a 10% gel with SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% BSA (BioFroxx; neoFroxx GmbH) or 5% milk (BD Biosciences) in TBS for 1 h at room temperature before incubating with primary antibodies (overnight at 4°C) and secondary antibodies (1 h at room temperature). 5% BSA was used to block membranes to assess p-YAP1; 5% milk was used to block membranes to assess other molecules (GALNT12, GAPDH, YAP1 and Lamin B1). The primary antibodies used were: GALNT12 (cat. no. K108365P; Beijing Solarbio Science & Technology Co., Ltd.), GAPDH (cat. no. GB15004; Wuhan Servicebio Technology Co., Ltd.), yes1 associated transcriptional regulator (YAP1; cat. no. ET1608-30; HUABIO), phosphorylated (p)-YAP1 (cat. no. ET1611-69; HUABIO) and Lamin B1 (cat. no. ET1606-27; HUABIO). The HRP-conjugated secondary antibodies (cat. no. GB23303; Wuhan Servicebio Technology Co., Ltd.) were used. Protein bands were visualized using ECL reagent (Wuhan Servicebio Technology Co., Ltd.) in ChemiDoc™ (BioRad Laboratories, Inc.). ImageJ/Fiji (2.15.0; Github) was used to measure the bands densitometry.

Nuclear proteins were extracted using the Nuclear Protein Extraction kit (cat. no. R0050; Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's protocols. Cells were washed with 1× PBS, and then cytoplasmic protein extraction reagent was added and the mixture was incubated on ice for 10 min. Cells were centrifuged (12,000 × g for 10 min at 4°C) and the supernatant was collected to obtain cytoplasmic proteins. Subsequently, nuclear protein extraction reagent was added to the remaining cell sediment. This mixture was incubated on ice for 10 min, and then the supernatant was collected after centrifugation (12,000 × g for 10 min at 4°C) to obtain nuclear proteins.

HT-1080 cells (5×10⁵) were seeded in 6-well plates before transfection. At 70% confluence, cells were transfected with 100 pmol siRNA against GALNT12 (siGALNT12-1 and siGALNT12-2) or non-targeting control siRNA (Table SII; TsingKe Biological Technology) using Lipo6000 (Beyotime Institute of Biotechnology) in Opti-MEM (Gibco; Thermo Fisher Scientific, Inc.). After 8 h at 37°C, high-glucose DMEM supplemented with 10% fetal bovine serum (Shanghai ExCell Biology, Inc.) was added. After 48 h, transfected cells were used for further experiments.

Cell viability was assessed using a CCK-8 assay. HT-1080 cells were seeded in 96-well plates (2×10³ cells/well) and transfected with GALNT12 siRNAs and negative control siRNAs. After transfection, 10 µl CCK-8 reagent (Beyotime Institute of Biotechnology) was added to each well and incubated for 2 h in the dark. Absorbance at 450 nm was measured using a SpectrumMax Plus384 microplate reader (Molecular Devices, LLC).

HT-1080 cells (2×10⁵ cells per well) were seeded in 6-well plates and allowed to adhere at 37°C for 24 h. Cells were incubated with EdU reagent (Wuhan Servicebio Technology Co., Ltd.), fixed with 4% paraformaldehyde, permeabilized with Triton X-100 and stained following the manufacturer's protocol. Nuclei were counterstained with 5 µg/ml Hoechst 33342 (Wuhan Servicebio Technology Co., Ltd.) for 15 min at room temperature. Cells were imaged using a Nikon inverted fluorescence microscope (Nikon ECLIPSE Ti2; Nikon Corporation).

HT-1080 cells were seeded in 6-well plates (3×10⁵ cells/well) and grown to 100% confluence. A sterile pipette tip was used to create vertical scratches across the cell monolayer and the cells were than cultured with fresh serum-free medium at 37°C. Images were captured at 0 and 24 h post-scratch using a Nikon inverted light microscope (Nikon ECLIPSE Ti2). Cell migration distance was judged by comparing the distance between the initial scratch (0 h) and the scratch at the 24-h observation point, which was measured using ImageJ/Fiji software (2.15.0; Github).

Transwell chambers (pore size, 8 µm; Corning, Inc.) were prepared with 500 µl serum-free DMEM in the upper chamber and 3×10⁵ HT-1080 cells/well. The lower chamber contained 500 µl DMEM with 20% FBS. After culturing at 37°C for 24 h, cells were fixed in 4% paraformaldehyde for 15 min, stained with 0.1% crystal violet for 10 min at room temperature, imaged under a light microscope (Nikon ECLIPSE Ti2) and counted.

GSEA (version 4.2.3, http://www.gsea-msigdb.org/gsea/index.jsp) was performed on the GSE2553 dataset to identify key enriched pathways. Normalized Enrichment Score (NES), P-value and false discovery rate (FDR) are the main observables for enrichment analysis (cut off values: NES >1, P<0.05, FDR <0.25).

GraphPad Prism (version 9.0; Dotmatics) was used for data analysis. Paired and unpaired Student's t-test, Fisher's exact test and one-way ANOVA (followed by Turkey's Honest Significant Difference test) were used to analyze statistical differences between groups. P<0.05 was considered to indicate a statistically significance difference.

To explore gene expression level changes in fibrosarcoma, the GSE24369 and GSE21124 public datasets from GEO were analyzed. A total of 308 and 578 differentially expressed genes (DEGs) in GSE24369 and GSE21124 were identified, respectively (P<0.05; log₂ fold change >1.5). Comparisons between these datasets identified 15 common DEGs (Fig. 1A). Among these, fatty acid binding protein 3, GALNT12, protein regulator of cytokinesis 1 and CDC28 protein kinase regulatory subunit 2 were highly expressed in tumors (Fig. 1B). Based on the statistical significance observed and the previous literature (19–23), GALNT12 was selected for further analysis. GALNT12 mRNA levels were significantly increased in the tumor groups in both datasets when compared with the control group (P<0.01; Fig. 1C). In the GSE21124 dataset, GALNT12 demonstrated significantly increased expression among GALNT family members GALNT7, GALNT10 and GALNT12 when compared with the control group (P<0.01; Fig. 1D). Immunofluorescent analysis demonstrated cytoplasmic localization of the GALNT12 protein in HT-1080 cells (Fig. 1E).

Analysis of both public and acquired patient data demonstrated increased GALNT12 mRNA and protein expression levels in fibrosarcoma. In the GSE2719 GEO dataset, GALNT12 mRNA expression levels were increased in tumor tissues compared with normal tissues (P<0.05; Fig. 2A). RT-qPCR results also demonstrated increased GALNT12 mRNA expression levels in HT-1080 cells compared with normal fibroblasts (P<0.05; Fig. 2B). Immunoblotting demonstrated increased GALNT12 protein expression levels in six fibrosarcoma samples compared with healthy tissue controls (Fig. 2C).

IHC staining demonstrated an increased expression of GALNT12 in fibrosarcoma tissues compared with adjacent healthy tissue (Fig. 3A and B). High GALNT12 expression was significantly associated with larger tumor size (P=0.002) and advanced T-stage (P=0.027) (Table I).

The present study investigated the impact of GALNT12 on the biological behavior of fibrosarcoma cells. siRNAs targeting GALNT12 were used to knock down GALNT12 expression levels in HT-1080 cells. The efficiency of transfection was confirmed with RT-qPCR analysis (Fig. 4A). Subsequently, the CCK-8 assay demonstrated reduced cell viability in the GALNT12 knockdown groups compared with the negative control group (Fig. 4B). Moreover, the EdU assay demonstrated a significant decrease in cell proliferation in the GALNT12 knockdown groups compared with the negative control (P<0.05; Fig. 4C and D).

To assess the influence of GALNT12 on cell motility, both wound healing and Transwell assays were used with transfected HT-1080 cells. The Transwell assay demonstrated a significant reduction in the number of cells traversing the chamber in the GALNT12 knockdown groups compared with the control group (P<0.0001; Fig. 5A and B). Additionally, the wound healing assay demonstrated a reduced migration in the GALNT12 knockdown groups compared with the control group (P<0.01; Fig. 5C and D).

Using the GSE2553 dataset from the GEO database, samples were categorized into high and low GALNT12 mRNA expression groups. Through GSEA, the YAP1 signaling pathway was identified as pivotal in fibrosarcoma pathogenesis (Fig. 6A). Following GALNT12 knockdown, the phosphorylation level of YAP1 increased compared with the negative control group (Fig. 6B). The nuclear protein extraction assay demonstrated reduced YAP1 nuclear localization in the siGALNT12-1 and siGALNT12-2 groups compared with the negative control (Fig. 6C and D). Furthermore, RT-qPCR analysis demonstrated a significantly decreased expression of YAP1 downstream target genes, including CYR61, AMOTL2 and BIRC5, compared with the corresponding negative control groups (P<0.05) (Fig. 6E). Based on these findings, it could be suggested that GALNT12 modulates the proliferation and migration capabilities of the fibrosarcoma cell line HT-1080 by influencing YAP1 nuclear localization (Fig. 7).