PYCR1 induces ferroptosis via the PI3K/Akt signaling pathway to regulate the proliferation and migration of osteosarcoma

Highlights

• •This study provides the first evidence that PYCR1 serves as a novel oncogenic driver in osteosarcoma, demonstrating through in vitro and in vivo experiments that PYCR1 significantly promotes osteosarcoma cell proliferation, migration, and other malignant biological behaviors.
• •It reveals the regulatory role of PYCR1 knockdown in inducing ferroptosis in osteosarcoma cells, thereby filling a gap in the understanding of the regulatory mechanisms underlying ferroptosis-mediated apoptosis in osteosarcoma.
• •The research elucidates that PYCR1 collaborates with the downstream gene COL1A1 and is regulated by the PI3K-AKT pathway to influence the proliferation, migration, and other biological behaviors of osteosarcoma, uncovering for the first time the interaction between PYCR1, extracellular matrix proteins, and classical oncogenic signaling pathways.
• •This study confirms the regulatory mechanism of the PI3K/Akt pathway in regulating ferroptosis in the HOS cell model of osteosarcoma.
• •The study systematically investigates the interactions among relevant genes and their functional mechanisms in osteosarcoma from multiple perspectives.

Abstract

Osteosarcoma treatment outcomes are compromised by increased drug resistance, distant metastasis, and tumor recurrence. While Pyrrolidine-5-carboxylic acid reductase 1(PYCR1) knockdown has been shown to inhibit tumor proliferation and migration in certain cancers. Its effects in osteosarcoma and regulatory mechanisms within osteosarcoma cells have not been explored. This study investigated the role of PYCR1 in osteosarcoma proliferation and its potential molecular mechanisms. The expression of PYCR1 was assessed in osteosarcoma tissues by immunohistochemistry and its correlation with clinical outcomes was determined. Subsequently, lentivirus-mediated knockdown and over expression of PYCR1 was achieved in osteosarcoma cells to evaluate its impact on proliferation, colony formation, and migration ability. The PI3K/Akt signaling pathway and its downstream effector COL1A1 were investigated using a combination of biomarker analysis, transcriptome sequencing, and co-immunoprecipitation assays. Subsequent silencing of COL1A1 induced phenotypic changes and confirmed tumorigenicity in vivo. The ferroptosis agonist Erastin and its specific antagonist Ferrostatin-1 (Fer-1) were combined with PYCR1 knockdown to explore their correlation with ferroptosis. By applying the pathway inhibitor LY294002, it was confirmed that the PI3K/Akt pathway is crucial for osteosarcoma proliferation. This study confirms that PYCR1 drives osteosarcoma cell proliferation and migration through three key mechanisms: regulating downstream genes, inhibiting ferroptosis, and activating the PI3K/Akt signaling pathway.

Graphical abstract

Introduction

Among adolescents and children, osteosarcoma is the most common malignant bone tumor [1]. In recent years, the prognosis for patients has significantly improved through the combination of neoadjuvant chemotherapy and limb-sparing surgery [2]. However, for cases involving metastatic lesions or chemotherapy resistance, the five-year survival rate remains below 20% [3]. Consequently, the therapeutic efficacy for osteosarcoma remains constrained by chemotherapy drug resistance [4], tumor recurrence [5], and distant metastasis [6]. Therefore, identifying novel oncogenic driver genes and potential therapeutic targets is crucial.

Pyrrolidine-5-carboxylic acid reductase 1 (PYCR1) [7] is localized to the mitochondrial matrix and exerts significant effects on cellular metabolic regulation, oxidative stress responses, and the progression of various pathological states. Although PYCR1 has been studied in certain cancers [8], its potential role in osteosarcoma remains experimentally unexplored.

Ferroptosis is an iron-dependent, lipid peroxidation-driven cell death mechanism [9]. Abnormal expression of its core regulatory molecules, such as Glutathione Peroxidase 4, is closely associated with osteosarcoma sensitivity to chemotherapy and impacts clinical treatment outcomes. GPX4 is a key negative regulator of ferroptosis by reducing lipid peroxides [10], while ACSL4 promotes ferroptosis by enriching long polyunsaturated fatty acids in phospholipids [11]. Therefore, they were selected as the target proteins for detection in this study on ferroptosis. Abnormal activation of the PI3K/Akt signaling pathway promotes tumor cell proliferation and suppresses apoptosis [12]. However, whether PYCR1 participates in the synergistic regulation of apoptosis and ferroptosis in osteosarcoma cells, and whether this process is mediated through the PI3K-Akt pathway, remains unreported. This study aims to identify downstream molecules and key signaling pathways significantly differentially expressed in ferroptosis through PYCR1 knockdown combined with transcriptome sequencing analysis. Experimental validation will investigate whether PYCR1 knockdown induces ferroptosis in osteosarcoma cells, regulates the PI3K/Akt signaling pathway, and affects the functions of downstream molecules. Based on the aforementioned research context and scientific questions, this study centers on PYCR1 gene knockdown to systematically investigate its biological functions and regulatory mechanisms in osteosarcoma.

Materials and methods

Clinical specimens and source data

Formalin-fixed, paraffin-embedded osteosarcoma tissue blocks were obtained from the Department of Pathology, The Third Affiliated Hospital of Southern Medical University, and were used for immunohistochemistry (IHC), immunofluorescence and Hematoxylin-Eosin staining. All patients provided their written informed consent before surgery scheduled between April 2022 and October 2024. This study was conducted with the approval of the Ethics Committee of The Third Affiliated Hospital of Southern Medical University. Tissue sections from osteosarcoma patients that had not received any radiotherapy or chemotherapy since the diagnosis of osteosarcoma were included in the study. Patients that had undergone radiation therapy or chemotherapy, and patients with tumors in other parts of the body were excluded.

Immunohistochemistry, fluorescence localization and Hematoxylin and Eosin (H&E) staining

Immunohistochemistry: Paraffin-embedded osteosarcoma sections were processed by baking (65 °C, 4 h), deparaffinization, and graded alcohol rehydration. Heat-mediated antigen retrieval was employed using a citrate buffer solution, followed by cooling to room temperature. Nonspecific sites were blocked prior to overnight incubation with the primary antibody at 4 °C. Finally, an HRP-conjugated secondary antibody was applied to the sections. DAB chromogen was applied (protected from light), and color development was microscopically monitored. Nuclear counterstaining was achieved with hematoxylin, followed by acid-alcohol differentiation, then dehydrated through graded alcohol solutions. After clearing with xylene, the coverslips were mounted using neutral resin. The slides were scanned the next day. A two-parameter scoring system was used, independently assessing stain intensity (0–3) and the percentage of immunoreactive cells (0–100%). Their product determined the final score: 0–4 (negative), 5–12 (positive). Correlations with clinical stage and distant metastasis were analyzed using these scores.

Fluorescence Localization: Cells were seeded on dedicated coverslips and allowed to grow until 70% confluence. They were then fixed with 4% paraformaldehyde at room temperature for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 5% bovine serum albumin at room temperature for 1 h. Add primary antibody mixture (PYCR1 and COL1A1) at concentrations specified in the antibody manual, incubate overnight at 4 °C. The next day, after warming to room temperature, add secondary antibody dropwise and incubate at room temperature in the dark for 1 h. Stain nuclei with DAPI for 5 min. Finally, mount slides with anti-fluorescence quenching mountant. Collect images under a laser confocal microscope to analyze protein colocalization.

H&E staining: 4-μm-thick sections were prepared, deparaffinized with xylene, rehydrated using a series of alcohol solutions, and then stained with hematoxylin and eosin. After staining, the sections were dehydrated and cleared, then mounted for microscopic imaging.

Cell culture

Human osteosarcoma cell lines (MNNG/HOS and U-2 OS) and normal human osteoblasts were obtained from Guangzhou Kefan Biotechnology Co., Ltd. (Guangdong, China) and were successfully identified. The osteosarcoma cells were cultured in DMEM-H medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin, and maintained at 37 °C in a humidified atmosphere containing 5% CO₂. Normal osteoblasts were cultured under the same medium conditions but maintained at 34 °C. All cell lines were authenticated and confirmed free of mycoplasma contamination.

Western blot and co-immunoprecipitation(co-IP)

Total protein extraction was performed with a dedicated lysis buffer system, supplemented with Phenylmethanesulfonyl fluoride and a protease inhibitor cocktail, in accordance with the manufacturer's guidelines (P0013, Beyotime Biotechnology Co., Ltd., Jiangsu, China). The homogenate was subjected to ice-cold lysis for 30 min and subsequently clarified by centrifugation. The determination of protein concentration in the supernatant was performed using the BCA assay. Finally, aliquots were denatured by boiling at 100 °C for 10 min in Laemmli buffer and stored at -20 °C. Before electrophoresis, the samples were mixed with 1/4 vol of 4 × loading buffer, heated to 100 °C for 5 min, and 10 μL of each sample was loaded per well (15-well format). Following electrophoresis and transfer, the membranes were blocked with a rapid sealing solution (Wuhan Servicebio Technology Co., Ltd., China) for 30 min at 26 °C. The membranes were probed with primary antibodies at 4 °C overnight. After washing, membranes were incubated with goat anti-mouse secondary antibodies, which were diluted in skim milk, for 1 h at room temperature. The target protein bands were finally visualized employing an enhanced chemiluminescence (ECL) system and analyzed. Clarified cell lysates were subjected to co-immunoprecipitation (co-IP) by incubation with designated antibodies overnight at 4 °C with constant stirring to isolate target protein complexes. The immunocomplexes were then coupled to protein A/G and after magnetic separation, the pellets were washed to remove nonspecific binding. Subsequently, the captured proteins are released by heating. The eluted immunoprecipitates were resolved by SDS-PAGE and detected by immunoblotting.

Quantitative PCR (qPCR)

Untreated normal cells and four groups of HOS cells infected with lentivirus were cultured for 72 h. Cells were lysed using TRizol reagent, chloroform layered to extract the supernatant and centrifuged with the addition of isopropanol to precipitate the RNA. Pellets were washed with 75% ethanol, and Diethyl Pyrocarbonate to solubilize the RNA, and the concentration was measured by spectrophotometry to evaluate the RNA quality. cDNA was synthesized using 2 μg of total RNA. Subsequent quantitative PCR amplifications were conducted in a reaction system containing SYBR Green Master Mix (Vazyme, Nanjing, China) on a real-time PCR instrument. Gene-specific primers, obtained from Beijing Qinke Biotechnology Co., Ltd., were utilized with GAPDH as the endogenous reference control. The relative quantification of gene expression was performed according to the 2^-ΔΔCt method.

PYCR1-(F) 5′-CGACATTGAGGACAGACACATTG-3′;

PYCR1-R: 5′-ACGACTGGAGTGTTGGTCATG-3′;

COL1A1-(F) 5′-CAG CCA ATC GCC AAG AGC-3′;

COL1A1-R: 5′-GCT CTT GGC GAT TGG CTG-3′;

GAPDH-(F) 5′-CAAGGTCATCCATGACAACTTTG-3′

GAPDH-R: 5′-GTCCACCCTGTTGCTGTAG-3′

CCK-8 assay

Log-phase cells from each experimental group were trypsinized, resuspended in complete medium, and counted. The cell density was adjusted to 2000 cells/well (100 μL/well) according to the growth rate and each treatment was repeated in 3 wells. The plate was agitated gently to ensure uniform distribution. After cell attachment (verified under a microscope), the 96-well plates were incubated. The CCK-8 reagent was added to each well 2 h prior to the endpoint assay. After 2.5 h of incubation, the plates were shaken for 3 min, using an enzyme marker absorbance was measured at 450 nm, and data was analyzed.

EdU incorporation assay

Log-phase HOS cells were seeded in 24-well plates (1 × 10⁵ cells/well).The cells were treated as indicated in triplicate. When confluence reached 40%, pre-warmed EdU (20 µM) was added at a 1:1 ratio (final concentration 10 µM) and incubated for 2 h at 37 °C according to the manufacturer's instructions (Beyotime Biotechnology). After fixation and permeabilization, cells were incubated with 100 µL/well of Click reaction mixture for 30 min at room temperature in the dark. Following washing, nuclei were stained with Hoechst 33342 for 10 min and images were captured using a fluorescence microscope. EdU-positive cells and total nuclei were counted to calculate the positive rate for statistical analysis.

Preparation of lentivirus and generation of stably transduced cells

Lentiviral constructs targeting the PYCR1 or COL1A1 gene were custom-synthesized and packaged by Shanghai JiKai Gene Co., Ltd. The lentiviral vector backbone was engineered to harbor a GFP (Green Fluorescent Protein)-PYCR1 or RFP (Red Fluorescent Protein)-COL1A1 fusion gene, along with a puromycin-selectable marker. The selectable markers for the two constructs were puromycin and neomycin, respectively. Prior to cellular transduction, the lentivirus titer was quantified via the limiting dilution assay, confirming a titer of ≥1 × 10⁸ TU/mL. The lentiviral preparations were subjected to safety certification by Shanghai JiKai Gene Co., Ltd, which confirmed the absence of contamination and verified their efficacy.

Analysis of apoptosis and reactive oxygen species(ROS) by flow cytometry

Cell apoptosis was detected using an Annexin V-APC/propidium iodide (PI) double-staining kit. Briefly, HOS cells were harvested with EDTA-free trypsin, washed twice with ice-cold phosphate-buffered saline (PBS), and counted. Subsequently, 1 × 10⁵ cells were resuspended in 500 μL of 1 × Annexin V binding buffer. The cells were then stained with 5 μL of Annexin V-APC and 5 μL of PI, followed by incubation for 15 min at room temperature in the dark. Samples were analyzed immediately by flow cytometry. Intracellular ROS levels were measured using a DCFH-DA probe (Beyotime Biotechnology) according to the manufacturer's instructions. Briefly, harvested cells were incubated with 1 mL of DCFH-DA working solution (diluted 1:1000 in serum-free medium) at 37 °C under 5% CO₂ for 20 min, with gentle inversion every 5 min during incubation. Subsequently, the cells were washed three times with fresh medium to remove excess probe, resuspended in 300 μL of medium, and immediately analyzed by flow cytometry.

Lipid peroxidation assay

Malondialdehyde (MDA) levels were measured to assess lipid peroxidation. Cells were harvested by trypsinization, washed twice with ice‐cold PBS, and counted. A total of 1 × 10⁶ cells were lysed in 300 μL of lysis buffer and centrifuged at 12,000 × g for 10 min at 4 °C. The resulting supernatant was collected, and MDA content was determined using a commercial MDA assay kit (Beyotime, S0131S, A221250116) according to the manufacturer’s protocol. Absorbance was read at 535 nm on a microplate reader, and results were automatically calculated.

Wound healing assay

A total of 10⁵ cells were inoculated in each well of a 6-well plate. After 3 days of incubation (when the cell confluence was greater than 90%), the monolayer of cells was scaped vertically with a 200 μL pipette tip guided by a straightedge. The wells were gently rinsed 2 to 3 times with serum-free medium and then incubated in medium containing 0.5% FBS for 24 h. Images were taken under a fluorescence microscope at 0 and 24 h after scratching and the extent of migration was determined by calculating the relative wound closure area. Healing (migration)rate (%) = (Wound area at 0 h–Wound area at 24 h)/Wound area at 0 h × 100%.

Transwell migration assay

Cell migration was assessed using Transwell chambers (24-well format, 8-μm pore size). After serum starvation in basal medium for 24 h, log-phase cells were harvested and resuspended at a density of 2.5 × 10⁵ cells/mL in serum-free medium. A 200 μL volume of cell suspension was added to the upper chamber, whereas the lower chamber was loaded with 500 μL complete medium containing 10% FBS as a chemoattractant. Following 24 h of incubation at 37 °C in 5% CO₂, non-migrated cells on the upper membrane surface were carefully removed with a cotton swab. Migrated cells on the lower surface were fixed with 4% paraformaldehyde for 20 min, washed with PBS, stained with 0.1% crystal violet for 10 min, and air-dried. Images were captured under an inverted microscope, and migrated cells were quantified in five random fields per well.

Colony formation assay

Once cells had reached 80% confluence, cells were rinsed with PBS and then counted. A total of 1000 cells was inoculated into each well of a 6-well plate containing 3 mL of complete medium. The cells were gently mixed and incubated for 7–to 10 days, changing medium after 3 days until colonies of ≥50 cells were visible under the microscope. The colonies were fixed with 4% paraformaldehyde for 20 min. The samples were subsequently washed with PBS, subjected to staining, and air-dried prior to microscopic imaging.

Transcriptome sequencing

Samples were sent to Shanghai JiKai Gene Co., Ltd. for sequencing. Sequencing groups included the shCtrl group (normal HOS cells) and the shPYCR1 knockdown group, each comprising three samples. RNA was extracted from cells using standard methods after achieving 80% confluence. RNA quality control followed, involving precise integrity assessment and mRNA enrichment. Following cDNA synthesis, library construction was performed through a series of enzymatic and clean-up steps, including end repair, A-tailing, adapter ligation, size selection, PCR amplification, and final purification, followed by strand-specific library preparation. Initial quantification and dilution were performed, and after library quality met expectations, quantitative real-time (qRT)-PCR was used for precise quantification of effective library concentration to ensure library quality. Significant differential gene expression across conditions was determined through appropriate statistical testing. Gene set enrichment analysis was performed using clusterProfiler (version 4.8.1). Enrichment was analyzed against GO, KEGG, Reactome, DO, and DisGeNET databases. An adjusted P-value < 0.05 was considered statistically significant.

Animal experiments

All animal experiments were conducted in accordance with protocols approved by the Animal Ethics Committee. Four-week-old male BALB/c nude mice were commercially sourced from Guangdong Zhiyuan Biological Laboratory Animal Co., Ltd. and were housed under a scientifically controlled environment. Fifteen mice were randomly divided into the shCtrl, shCOL1A1, and shCOL1A1+shPYCR1 groups, and were injected subcutaneously with a suspension of HOS cells (10⁶/200 μL) to establish the tumor model. Tumor growth was observed, and tumor volume was calculated and recorded weekly. Following the 4-week experimental period, all mice were humanely euthanized, after which tumor tissues were excised and subjected to IHC analysis. This study utilized pentobarbital for static injection anesthesia in nude mice, The compound was administered by tail vein injection at a dose of 100 mg/kg body weight. Within 3 min post-injection, the disappearance of pulse, respiration, and corneal reflexes was observed.

Transmission electron microscopy

Following cell collection, cells were washed with PBS and then fixed with prechilled fixative. The resulting pellets were refrigerated at 4 °C for 6 h for post-fixation, followed by immediate transfer to sodium dimethylarsenate buffer. After 2 h of fixation at 4 °C, pellets were washed three times with sodium dimethylarsonate buffer, and then fixed for another 2 h. A graded series of acetone-resin mixtures was used for dehydration and permeation: 1:1 for 2 h; then 1:2 for 4 h; and finally pure resin overnight. This was followed by embedding and polymerization/hardening. The specimens were trimmed to an appropriate size and ultrathin sections were prepared at 60 nm thickness. Preparations were stained, and prepared for electron microscopy observation, and digital photography.

Statistical analysis

All statistical analyses were performed using GraphPad Prism (v.9.5.0) and SPSS (v.24.0). The normality of data distribution was assessed prior to the selection of statistical methods. Nonparametric tests were applied when the data deviated from a normal distribution. Multiple group comparisons were conducted by one-way Analysis of Variance, whereas differences between groups in cell phenotype assays and subcutaneous tumorigenicity experiments were evaluated using Student’s t-test or ANOVA, as appropriate. Rank sum tests were applied to analyze PYCR1 expression differences between osteosarcoma tissues and adjacent normal tissues. Correlations between PYCR1/COL1A1 expression and tumor biological characteristics were also assessed. Statistical significance was set at P < 0.05. Significance levels are denoted as *P < 0.05, **P < 0.01, and ***P < 0.005.

Results

PYCR1 demonstrated significant overexpression in osteosarcoma, which was positively correlated with the pathological stage of the disease

IHC revealed significantly elevated PYCR1 expression in osteosarcoma specimens relative to tissues adjacent to the tumor and normal bone tissue. (Fig. 1A). Furthermore, PYCR1 was localized in the cytoplasm of osteosarcoma cells (Fig. 1B). Ki67 staining confirmed the malignant nature of osteosarcoma and HE staining revealed hyperchromatic nuclei, disorganized cell arrangement, and marked atypia in the osteosarcoma tissue (Fig. 1C,D). The study shows that PYCR1 expression was not significantly affected by patient sex, age, T-cell infiltration, or lymph node metastasis (Supplementary Table S1). In clinical pathological sections of osteosarcoma, tumor characteristics were positively correlated with PYCR1 expression and were associated with pathological staging and malignancy grade (Supplementary Table S2).

Fig. 1.

Exploration of PYCR1 expression in osteosarcoma. (A) Immunohistochemistry reveals that PYCR1 expression is significantly higher in osteosarcoma than in adjacent tissue and normal tissue. (B) Immunofluorescence indicates that PYCR1 localizes in the cytoplasm. Scale bars:50 μm. (C) Ki67 staining showed a significant difference between osteosarcoma tissue and tumor-adjacent tissue. (D) HE staining reveals that osteosarcoma cells exhibit marked atypia, deep nuclear staining, and disorganized arrangement. (E) Brightfield and GFP fluorescence images of HOS and U2OS osteosarcoma cells after PYCR1 knockdown. (F) Western blotting revealed high PYCR1 expression in osteosarcoma HOS and U-2OS cells in comparison to normal osteoblasts. Subsequent screening of three lentiviral constructs identified shPYCR1-1 and shPYCR1-3 as effective knockdown sequences, as confirmed by western blotting. (G) Quantitative real-time PCR (qPCR) further confirmed that PYCR1 was successfully knocked down by shPYCR1-1 and shPYCR1-3 in HOS cells. In all figures, statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

Based on these findings, PYCR1 was selected as the target gene for further investigation. Western blot analysis revealed that PYCR1 expression levels were higher in both osteosarcoma cell lines (HOS and U-2OS) than in normal osteoblasts (Fig. 1F). A lentiviral vector carrying green fluorescent protein (GFP) was employed to knock down PYCR1, allowing for discrimination from untransfected osteosarcoma cells (Fig. 1E). To evaluate the knockdown efficiency, an empty vector was used as a negative control. Western blotting and qPCR analyses confirmed successful knockdown of two independent targets (Fig. 1G).

Validation of PYCR1 function in osteosarcoma cells

Western blot analysis confirmed two effective short hairpin RNA (shRNA) targeting sequences against PYCR1, designated shPYCR1-1 and shPYCR1-3, which were subsequently employed for dual-target validation. CCK-8 assays demonstrated that PYCR1 knockdown significantly suppressed the proliferation of osteosarcoma cells (Fig. 2A). Consistently, wound healing and Transwell assays revealed that PYCR1 depletion impaired both the horizontal and vertical migration capacities of osteosarcoma cells (Fig. 2B, C). Furthermore, colony formation assays performed in triplicate using HOS cells showed that PYCR1 knockdown markedly reduced clonogenic potential (Fig. 2D). Flow cytometry analysis further revealed that PYCR1 knockdown led to a significant increase in the apoptotic rate of osteosarcoma cells (Fig. 2F). Collectively, these findings indicate that PYCR1 plays a pro-tumorigenic role in osteosarcoma cells.

Fig. 2.

Role of PYCR1 in osteosarcoma cells. (A) Dual-target validation of PYCR1 knockdown efficiency. The CCK-8 proliferation assay demonstrated a significantly reduced proliferation rate in the shPYCR1-1 group. (B) Wound healing assay results for HOS and U-2OS cells. The wound-healing ability was significantly reduced in the shPYCR1-1 group (P < 0.05). (C) Migration assays revealed a markedly diminished migration ability in the shPYCR1-1 group, showing significant differences compared to the negative control and shPYCR1-3 groups. (D) Colony formation assay revealed that PYCR1 knockdown significantly decreased the clonogenic ability of HOS cells. (E) Apoptosis assays demonstrated more pronounced apoptotic phenomena in the shPYCR1-1 group.

Effects of PYCR1 overexpression on osteosarcoma

Following lentiviral transduction of osteosarcoma HOS cells with PYCR1 overexpression vectors, western blotting was performed to verify the expression efficiency (Fig. 3A). PYCR1 expression was markedly elevated in the overexpression group but was barely detectable in the knockdown group. Functional assays revealed that PYCR1 overexpression significantly accelerated HOS cell proliferation compared with controls, as determined by CCK-8 assays (Fig. 3B). Wound healing assays further demonstrated that PYCR1 promotes horizontal migration of osteosarcoma cells (Fig. 3C). Moreover, Transwell migration and colony formation assays showed that PYCR1 enhances both vertical migration and clonogenic capacity (Fig. 3D, E).

Fig. 3.

Effects of PYCR1 Overexpression on HOS cells. (A) PYCR1 overexpression efficiency was confirmed by Western blot, and knockdown results were consistent with previous validation. (B) The CCK-8 proliferation assay demonstrated a significantly increased HOS cells proliferation rate in the PYCR1-overexpressing group. (C) Migration assays revealed significantly enhanced cell migration capacity in the PYCR1-overexpressing group, showing significant differences from the negative control group. (D) Colony formation assay in triplicate revealed that PYCR1-overexpressing group increased HOS cells colony ability compared to the control group. (E) Transwell assays in triplicate demonstrated enhanced HOS cells vertical migration ability following PYCR1 overexpression.

Identification of major signaling pathways and downstream genes and validation of protein interactions

Based on the validation of knockdown efficiency for the target PYCR1, we selected HOS cells from the shPYCR1-1 group for transcriptome sequencing. The RNA sequencing results (GSA-Human: HRA014926) yielded a gene enrichment profile. The Volcano plot revealed 531 up-regulated genes and 189 downregulated genes (Fig. 4A,B). The PI3K/Akt signaling pathway may serve as a key downstream pathway of PYCR1 (Fig. 4C). To investigate the potential biological functions and pathways associated with PI3K/Akt signaling, we performed enrichment analysis using the clusterProfiler package. A total of 21 differentially expressed genes were subjected to enrichment analysis: 16 upregulated (e.g., ITGA11, SYK, COL1A1) and 5 downregulated (e.g., NR4A1, PPP2R5B, EFNA2).Through literature review and data analysis from major gene databases (Genecards and TNMplot), COL1A1 was selected as a downstream target for further investigation. The presence of yellow in certain microregions under immunofluorescence suggests a potential interaction between PYCR1 and COL1A1 (Fig. 4D), and co-IP experiments further supported the likelihood of a protein-protein interaction.

Fig. 4.

Signaling pathway and downstream gene screening. (A) Transcriptome sequencing results showing the gene collection map. (B) Volcano map showing 531 up-regulated genes and 189 down-regulated genes. (C) Sequencing revealed downstream genes in a pooled histogram; the pathway having the most enriched genes was considered most relevant. (D)Immunofluorescence staining revealed that PYCR1 (red) and COL1A1 (green) were enriched in certain perinuclear regions, appearing yellow, suggesting a possible interaction between the two proteins. (Scale bar: 50 μm). (E)Reciprocal co-IP assays showed that PYCR1 interacted with COL1A1 in both forward and reverse immunoprecipitations, with no specific signals detected in the IgG controls. These findings indicate a biological association between PYCR1 and COL1A1, and PYCR1 knockdown significantly altered COL1A1 protein levels. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Reciprocal co-IP assays revealed that PYCR1 was present in the COL1A1 immune complex during forward immunoprecipitation, while COL1A1 was conversely detected in the PYCR1 immune complex in the reverse assay. Input controls confirmed the expression of both proteins in cell lysates, and no specific bands were observed in the IgG control groups, ruling out non-specific binding. Taken together, these findings provide evidence for a biochemical interaction between PYCR1 and COL1A1, suggesting that they form a complex and may constitute a novel regulatory axis for collagen synthesis. In support of this, knockdown of PYCR1 led to marked alterations in COL1A1 protein levels (Fig. 4E).

Knocking down COL1A1 inhibits osteosarcoma cell proliferation; knocking down both PYCR1 and COL1A1 results in a more significant inhibition of osteosarcoma cells and promotes apoptosis

COL1A1 is highly expressed in osteosarcoma (Fig. 5A) and was highly expressed in HOS and U2OS cells (Fig. 5B). Wound healing assay revealed that COL1A1 influenced the lateral migration of osteosarcoma cells (Fig. 5C). Knockdown of COL1A1 significantly attenuated these oncogenic phenotypes. In order to verify the involvement of the PYCR1/COL1A1 gene axis and the interaction function of the 2 genes, the expression of both PYCR1 and COL1A1 was knocked down to clarify the downstream effects. After PYCR1 is knocked down in HOS cells using a lentivirus, the cells exhibit green fluorescence (GFP). After COL1A1 is knocked down, they exhibit red fluorescence (RFP). When both genes are knocked down in a single cell, the fluorescent colors overlap to produce yellow (Fig. 5D)and verified by western blotting (Fig. 5E). Compared to single-gene knockdown, dual knockdown of PYCR1 and COL1A1 significantly suppressed osteosarcoma cell malignancy, as evidenced by reduced proliferation and migratory capacity in HOS cells (Fig. 5F, G). Moreover, flow cytometry analysis showed that dual knockdown markedly elevated ROS levels and induced apoptosis (Fig. 5H). These results indicate that PYCR1 and COL1A1 synergistically promote the growth of osteosarcoma cells.

Fig. 5.

Role of COL1A1 in osteosarcoma. (A) Immunohistochemistry results indicate high expression of COL1A1 in osteosarcoma tissue. (B) Western blotting analysis reveals high expression of COL1A1 in osteosarcoma HOS and U-2OS cell lines, whereas normal tissues show lower expression. COL1A1 was successfully knocked down in one group using lentivirus vectors. (C) Wound healing ability is significantly impaired in the shPYCR1-1 group compared with shCtrl group (P < 0.005). (D) When PYCR1 knockdown (GFP) and COL1A1 knockdown (RFP) coexist in the same cell, the cell’s fluorescence turns yellow. Images showed that the vast majority of cells exhibited dual knockdown of PYCR1 and COL1A1. (E) Validation of the knockdown efficiency by Western blot. (F) CCK-8 assay revealed that after simultaneous knockdown of PYCR1 and COL1A1, proliferative ability was significantly reduced, exhibiting worse performance than after COL1A1 knockdown alone. (G) After simultaneous knockdown of PYCR1 and COL1A1 genes, the migration ability of HOS cells is significantly reduced. (H) Flow cytometry revealed that knocking down both PYCR1 and COL1A1 genes simultaneously resulted in more apoptotic cells than knocking down either gene individually. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

In vivo tumorigenicity experiments showed that after double knockdown, the subcutaneous tumors were smaller in size and weight after 4 weeks than those in the single gene knockdown group and the empty vector group. The three groups of BALB/c nude mice were sacrificed after 4 weeks (Fig. 6A), and the tumors were excised (Fig. 6B). The tumors were the largest in the shCtrl group, followed by those in the shCOL1A1 group, whereas the smallest were in the shCOL1A1 + shPYCR1 group (Fig. 6D). The results were consistent in terms of tumor weight (Fig. 6C), with significant differences across all three groups. IHC analysis confirmed that the COL1A1 knockdown group yielded negative results, no significant positive staining was observed for either PYCR1 or COL1A1 in the double-knockdown group (Fig. 6E).

Fig. 6.

Subcutaneous tumor formation was used to verify the effect of gene knockdown. (A) Tumor conditions of immunodeficient mice 4 weeks after subcutaneous tumor formation. (B) Gross appearance of subcutaneous tumors. The double gene knockdown group exhibited the smallest tumor volumes. (C) Comparison of tumor weights revealing differences between groups. (D) Comparison of tumor volumes. (E) Immunohistochemical analysis of tumor specimens following simultaneous knockdown of upstream and downstream genes revealed decreased levels of protein PYCR1 and COL1A1. Knockdown of COL1A1 did not significantly alter protein PYCR1 expression levels.

PYCR1 knockdown sensitizes osteosarcoma cells to ferroptosis

Previous studies have confirmed the oncogenic role of PYCR1 in osteosarcoma. Transcriptomic sequencing revealed an enrichment of the ferroptosis pathway. To further investigate the underlying mechanisms, To elucidate the regulatory relationship between PYCR1 and ferroptosis in HOS cells, we assessed ROS levels and the expression of GPX4 and ACSL4 as key readouts. The green fluorescence inherent to lentiviruses significantly interfered with the C11-BODIPY-based lipid peroxide assay, making it unsuitable for this experiment. Therefore, additional methods, such as electron microscopy, were subsequently employed for validation. This study established four groups to validate ferroptosis, including the shPYCR1 group, shPYCR1+ Erastin group, shPYCR1 + Fer-1 group, and the empty vector control group (shCtrl).Based on literature references and our experimental validation, along with the solubility in < 0.1% DMSO and dose verification, the experimental concentrations were determined to be 2.5 μM for Erastin and 1 μM for Fer-1. The shCtrl group and the shPYCR1+Fer-1 group both exhibited high cell viability and fluorescence intensity, whereas the shPYCR1 group showed relatively lower levels. The shPYCR1+Erastin group exhibited extremely poor cell viability, with a large number of cells in suspension (Fig. 7A). The results of the EdU incorporation Assay confirmed the above conclusions (Fig. 7D). In addition, MDA levels in the shPYCR1+Erastin group were significantly higher than those in the other three groups, indicating that the induction of ferroptosis promotes apoptosis in HOS cells (Fig. 7B). Western blotting analysis revealed that In the Erastin-treated group, GPX4 expression was significantly reduced compared to the shCtrl group, with a more pronounced trend than in the shPYCR1 group; GPX4 expression increased following Fer-1 intervention. In contrast, ACSL4 expression was elevated in both the shPYCR1 and Erastin-treated groups compared to the shCtrl group (Fig. 7C). The ROS testing by Flow Cytometry showed that ROS levels in cells treated with Erastin increased significantly, and the curve shifted significantly to the right (Fig. 7E). These findings indicate that the ferroptosis pathway is one of the mechanisms underlying apoptosis in osteosarcoma cells and can be restored by Fer-1.

Fig. 7.

Validation of PYCR1-mediated HOS cells apoptosis and ferroptosis. (A) Cell viability and fluorescence intensity was higher in shCtrl and shPYCR1+Fer-1 group. (B) MDA assays revealed elevated MDA levels in the shPYCR1 group, with a more pronounced increase following the addition of Erastin. This effect was reversed by Fer-1. (C) Western blot analysis showed that GPX4 expression was significantly reduced in the shPYCR1 + Erastin group, followed by the shPYCR1 group. In contrast, ACSL4 levels were markedly elevated following PYCR1 knockdown and Erastin treatment. However, protein expression exhibited an opposite trend in the shPYCR1 + Fer-1 group and the shCtrl group. (D) EdU proliferation assay results showed that PYCR1 knockdown and Erastin treatment significantly inhibited HOS cell proliferation, whereas Fer-1 completely reversed this effect, promoting the restoration of cell proliferation to levels close to those of the control group. (Scale bar: 200 μm) (E) Flow cytometry ROS detection across all four groups indicated significantly elevated ROS levels and a marked rightward shift in the shPYCR1and shPYCR1+Erastin group. (F) Mitochondrial changes observed under electron microscopy (magnification:40,000x). (G) Western blot analysis revealed that COL1A1 expression in the OE-PYCR1 group was slightly higher than that in the shPYCR1 group but remained lower than that in the control group. GPX4 expression levels followed a similar pattern. (H) CCK-8 assays revealed that osteosarcoma cells in the OE-PYCR1 group exhibited the fastest proliferation, while those in the shPYCR1 group showed the slowest growth. No significant difference in proliferation rate was observed between the OE-PYCR1+shCOL1A1 group and the control group.

Transmission electron microscopy revealed that mitochondria in osteosarcoma cells exhibited an oval or rod-like shape with well-defined outlines and abundant cristae in both the empty vector control group and the shPYCR1+Fer-1 group. In contrast, shPYCR1-treated mitochondria showed reduced numbers and disorganized cristae structures. Following Erastin addition, cells exhibited swelling, membrane rupture, leakage of contents, and blurred cristae structures (Fig. 7F). This finding provides additional evidence for ferroptosis. We further examined the effects of PYCR1 overexpression on the expression of downstream genes, including GPX4 and COL1A1. COL1A1 expression in the OE-PYCR1 group was slightly higher than that in the shPYCR1 group but remained lower than that in the control group. GPX4 expression exhibited a similar trend (Fig. 7G). CCK-8 assay results revealed that osteosarcoma cells in the OE-PYCR1 group displayed the fastest proliferation rate, whereas those in the OE-PYCR1+shCOL1A1 group showed a proliferation rate comparable to that of the shCtrl group. In contrast, the slowest cell growth was observed in the shPYCR1 group (Fig. 7H). These findings further support the involvement of PYCR1 in ferroptosis and its upstream regulatory role within the PYCR1-COL1A1 gene axis. There may be compensatory mechanisms or pathways involved in COL1A1 expression.

The PI3K/Akt signaling pathway regulates ferroptosis and the PYCR1/COL1A1 gene axis in osteosarcoma cells.

In this set of experiments, we chose to intervene using LY294002, an inhibitor of the PI3K/Akt signaling pathway. EdU staining and Transwell assays confirmed that LY294002 affected the proliferation and migration capacity of HOS cells (Fig. 8A, B). Western blot analysis revealed that, compared with the shCtrl group, the shPYCR1 group exhibited reduced GPX4 levels and increased ACSL4 expression. However, these trends disappeared upon inhibition of the PI3K/Akt pathway, suggesting that this signaling pathway regulates ferroptosis. Furthermore, following inhibition of this signaling pathway, the expression of COL1A1 and its downstream molecules, p-Akt and Akt, all decreased. These results indicate that this signaling pathway exerts a positive regulatory effect on the expression of downstream molecules, leading to downregulation of these proteins (Fig. 8C). ROS levels in the shPYCR1 group were significantly higher than those in the shCtrl group. However, the ROS curve shifted more markedly to the right in the LY294002 + shPYCR1 group, with a significant increase in ROS levels (P < 0.05) (Fig. 8D). Malondialdehyde (MDA) assays further confirmed that inhibition of the PI3K/Akt pathway led to increased apoptosis in HOS cells and elevated MDA levels (Fig. 8E). In summary, these findings suggest that the increased apoptosis rate following pathway inhibition is attributable not only to the suppression of oncogenes but also to the induction of ferroptosis. The PI3K/Akt signaling pathway regulates ferroptosis and the PYCR1/COL1A1 gene axis in osteosarcoma cells.

Fig. 8.

Mechanism of regulation of the PI3K/Akt signaling pathway. (A) Upon inhibition of the PI3K/Akt pathway, the proliferation of osteosarcoma HOS cells decreased significantly. (Scale bar: 200 μm) (B) Migration assays revealed that LY294002 treatment markedly impaired the migratory capacity of HOS cells. (C) Western blot results showed that, compared to the shCtrl group, GPX4 levels showed a downward trend in the shPYCR1 group, while ACSL4 increased. However, these trends stopped following inhibition of the PI3K/Akt pathway. Furthermore, COL1A1, as well as p-Akt and Akt, exhibited downregulated expression. (D) Compared to the control group, ROS levels significantly increased after the addition of LY294002. (P < 0.05). (E) Treatment with LY294002 significantly increased MDA levels in osteosarcoma cells, indicating enhanced lipid peroxidation.

Discussion

Tumor cells exhibit enhanced metabolic activity, requiring substantial energy and employ multiple metabolic pathways supported by numerous enzymes. PYCR1 is overexpressed in some tumors and possesses oncogenic properties [13]. However, its role in osteosarcoma remains unexplored. Immunohistochemical analysis demonstrated that PYCR1 not only promotes osteosarcoma cell proliferation but is also significantly elevated in clinical osteosarcoma tissues [14]. Following PYCR1 knockdown via lentiviral transfection, cell proliferation, colony formation, and migration abilities all decreased. Overexpression of the target gene can reverse the associated biological function.

Transcriptome sequencing revealed PYCR1 involvement in multiple pathways, including the PI3K/Akt pathway [15] and the ferroptosis pathway [16]. Among the genes enriched in the PI3K/Akt pathway, previous studies have reported that interleukin-6 (IL-6) [17]and spleen tyrosine kinase (SYK) can activate and upregulate this signaling pathway [18]. In addition, Integrin Subunit Alpha 10(ITGA10) and Integrin Subunit Alpha 11(ITGA11), which belong to the integrin superfamily, are also involved in the activation of PI3K [19]. However, molecules such as nuclear receptor subfamily 4 group A member 1 (NR4A1) dephosphorylate and inactivate Akt [20], thereby inhibiting the PI3K/Akt signaling pathway. COL1A1 encodes type I collagen. It may act as a microenvironmental signal, indirectly influencing the balance of positive and negative regulation of the PI3K/Akt pathway through matrix-cell signaling and extracellular matrix remodeling.

Given PYCR1 abundance and the limited evidence available in the literature, we focused our investigation on the PI3K/Akt signaling pathway and its downstream gene COL1A1. Activation of the PI3K/Akt pathway is important to osteosarcoma proliferation and is associated with metabolic reprogramming in multiple cancers [21]. Its clinical relevance lies in the fact that PYCR1-driven proliferation and metastasis align with findings in other malignancies [22]. Although the function of COL1A1 in osteosarcoma remains unclear, IHC in this study confirmed its high expression in osteosarcoma tissues. Lentiviral knockdown of COL1A1 significantly inhibited biological functions including proliferation and colony formation in osteosarcoma cells [23], indicating its critical role in tumor progression. Co-IP experiments and fluorescence localization revealed a potential link between PYCR1 and COL1A1 protein. Given COL1A1′s link to osteoporosis [24] and public data TNM plot [25] and GeneCards [26]), suggesting a functional interaction, we propose a PYCR1-COL1A1 functional axis.

We have preliminarily validated the hypothesis that PYCR1 drives osteosarcoma progression through the PI3K/Akt pathway and induces ferroptosis to promote osteosarcoma apoptosis [27]. Knockdown of PYCR1 could sensitize cells to ferropotosis [28]. This is consistent with the report by Stockwell, which indicated that inhibition of the PI3K/Akt axis enhanced the induction of iron oxidation in cancer cells [29]. The change in the double knockdown phenotype was significantly stronger than that of single knockdown, suggesting the existence of linear dependence or functional superposition of the signaling pathway. When PYCR1 is knocked down, COL1A1 expression decreases, while overexpressed, COL1A1 expression increases; this finding supports the likelihood of a linear relationship between them. COL1A1 has also been studied more extensively in tumors. Other studies have determined that COL1A1 mediates tumor-stromal crosstalk through the ANGPTL4/SDC4 axis in gastric cancer [30]. Altogether, these data confirm that COL1A1 is a downstream effector of PYCR1 in the PI3K/Akt pathway, which drives the development of osteosarcoma and may represent a potential therapeutic target [31]. With declining sequencing costs, improved tools, and effective analysis of complex large-scale data [32], it has become possible to effectively describe the entire genome in a clinically relevant timeframe. This capacity has improved tumor treatment and disease control [33].

Aberrant gene fusions are widely prevalent in cancer genomes, among which a small fraction act as a disease driver and the remainder are nonspecific passenger fusion genes [34]. This study focused on the results of knockdown experiments to address tumor drug resistance [35], combined with functional analysis of background expression. PYCR1 presented significantly higher baseline expression in osteosarcoma cells, a finding that has been well-established. Knockdown experiments, which simulated the therapeutic scenario of “clinically targeting this gene for inhibition” hold greater clinical relevance. Overexpression of exogenous genes may induce cytotoxicity or activate compensatory pathways, potentially amplifying abnormal states and yielding false-positive results that compromise clinical screening [36]. Knockdown-induced phenotypic (proliferation, migration) and mechanistic changes robustly validate the functional effects, while the conservation of stress signaling pathways suggests an evolutionarily conserved regulatory mechanism. That prevents induction of gene mutations, plays an extremely vital role in maintaining cellular homeostasis [37]. Wu et al. [38] employed a similar approach by knocking out CENP-N expression to inhibit the Akt/mTOR pathway, and thereby enhanced the sensitivity of nasopharyngeal carcinoma to radiotherapy. In the study, PI3K/Akt signaling pathway acts as a valve, regulating gene expression and the ferroptosis. We will conduct in-depth investigations to explore their deeper relationships.

In conclusion, PYCR1 was identified as a novel gene-controlled target point for osteosarcoma. Its mechanism of action involves promoting ferroptosis and blocking the PI3K/Akt signaling axis. Based on the findings of this study, developing a PYCR1 gene diagnostic kit for osteosarcoma holds potential for the clinical screening of the disease. It may also be utilized for drug optimization, regulating ferroptosis to induce osteosarcoma shrinkage or control tumor proliferation, thereby achieving clinical therapeutic effects. These findings open new strategic avenues for osteosarcoma treatment.

Ethical approved and informed consent statement

All procedures involving human subjects in this study were conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Third Affiliated Hospital of Southern Medical University (Approval no.: 2025-ER-039).

Animal studies was approved by Guangzhou Lai'an Technology Animal Ethics Committee (no. G2025018).

Consent for publication statement

All authors have reviewed and approved the final version of the manuscript and explicitly consent to its publication. There are no undisclosed conflicts that could influence the research or its presentation.

Funding information

This work was supported by Guangdong Basic and Applied Basic Research Foundation (Approval no. 2022A1515012479); President Foundation of The Third Affiliated Hospital of Southern Medical University (Approval nos. YM202209, YP202216)

CRediT authorship contribution statement

Jianhua Hu: Project administration, Methodology, Investigation, Data curation, Conceptualization. Yiyuan Huang: Software, Formal analysis. Haoming Chen: Software, Data curation. Zehao Xie: Software. Han Yan: Resources. Haomiao Li: Writing – review & editing, Resources. Runguang Li: Writing – review & editing, Visualization, Validation, Supervision, Investigation, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare no conflicts of interest.

Data availability

The datasets are available from the corresponding author upon reasonable request. All data used in this research comply with relevant ethical guidelines and institutional regulations.