STRA6-mediated ferroptosis and its potential association with antioxidant stress pathways in glioblastoma

Summary

Glioma is the most common malignant tumor of the central nervous system, but the role of stimulated by retinoic acid 6 (STRA6) in its progression remains unclear. We established STRA6-knockdown and STRA6-overexpressing glioma cell lines to investigate their effects on malignant behavior and ferroptosis and validated the findings in a subcutaneous allograft model. STRA6 was upregulated in glioma tissues and associated with poor overall survival. Silencing STRA6 inhibited glioma cell proliferation, migration, and invasion; increased Fe²⁺ and malondialdehyde levels; reduced glutathione levels; and induced ferroptosis, whereas STRA6 overexpression exerted opposite effects. Ferrostatin-1 and deferoxamine attenuated ferroptosis induced by STRA6 knockdown, while Erastin reversed the effects of STRA6 overexpression. Collectively, these findings suggest that STRA6 promotes glioma progression by suppressing ferroptosis through the regulation of cellular antioxidant stress-related pathways, highlighting STRA6 as a potential therapeutic target for glioma.

Graphical abstract

Highlights

• •STRA6 is a key regulator of ferroptosis in glioblastoma
• •STRA6 depletion suppresses malignant phenotypes of glioblastoma cells
• •STRA6 is associated with antioxidant stress-related signaling in glioblastoma
• •STRA6 may serve as a promising therapeutic target for glioblastoma

Molecular biology; Cancer

Introduction

Gliomas represent the most common primary tumors of the central nervous system (CNS), comprising approximately 30%–40% of all primary brain tumors.¹ Recent epidemiological data show a rising global incidence, especially in elderly populations.² Recognized risk factors include genetic susceptibility, radiation exposure, and environmental influences.³ Histological and biological characteristics classify gliomas into grades I–IV, with grade I tumors demonstrating low malignancy and favorable outcomes, while grades II–IV exhibit progressively greater invasiveness and recurrence risk.⁴ Nevertheless, traditional histopathological classification fails to fully capture molecular heterogeneity, limiting its accuracy in predicting therapeutic response.⁵ The 2021 World Health Organization (WHO) classification of CNS tumors therefore incorporates molecular markers—including IDH mutations, 1p/19q codeletion, TERT promoter mutations, and MGMT methylation—to improve diagnostic precision and prognostic evaluation.^6,7 Glioblastoma (GBM), a grade IV glioma, constitutes the most malignant subtype.⁸ Its aggressive and heterogeneous nature enables tumor cells to infiltrate brain parenchyma along white matter tracts and perivascular spaces, rendering complete resection nearly impossible and recurrence inevitable.^9,10,11 Even with standard treatment involving maximal safe resection, radiotherapy, and temozolomide chemotherapy (the Stupp regimen), median survival remains only 14–18 months, and the 5-year survival rate is below 5%.¹² This poor prognosis relates to genomic instability, intratumoral heterogeneity, dysregulated signaling pathways, impaired DNA repair, and an immunosuppressive microenvironment.^13,14,15 Although advances in targeted therapy, drug delivery, immunotherapy, and metabolic modulation continue to be investigated,¹⁶ clinical benefits have been modest. Consequently, elucidating novel molecular mechanisms and therapeutic targets is critically needed. Recent studies in ferroptosis regulation,¹⁷ metabolic reprogramming,¹⁸ and immune remodeling of the tumor microenvironment are providing new insights and potential avenues for glioma therapy.³

Ferroptosis is an iron-dependent form of programmed cell death characterized by the toxic accumulation of lipid peroxides, which disrupts the integrity of the cell membrane.¹⁹ It exhibits unique morphological features, such as shrunken mitochondria with diminished cristae and increased membrane density that distinguish it from apoptosis or necrosis.²⁰ Driven primarily by iron-mediated lipid peroxidation rather than classical apoptotic pathways, ferroptosis typically involves membrane rupture, cellular content leakage, and irreversible damage.²¹ Within the mitochondrial electron transport chain, respiratory activity generates reactive oxygen species (ROS), while polyunsaturated fatty acids (PUFAs) serve as key substrates whose peroxidation yields toxic lipid peroxides that destabilize cellular membranes.^22,23 The impairment of antioxidant systems, particularly the inactivation of glutathione peroxidase 4 (GPX4), further diminishes the clearance capacity for lipid peroxides and accelerates ferroptotic death.²⁴ Excessive ROS and hydroxyl radicals (·OH) produced via the Fenton reaction additionally amplify lipid peroxidation.²⁵ Ferroptosis participates in diverse pathological conditions, including neurodegenerative diseases, ischemia-reperfusion injury, and tumor progression, where it notably contributes to GBM advancement, immune evasion, and therapeutic resistance.^26,27 Interventions targeting ferroptosis pathways—such as GPX4 inhibition, system Xc⁻ blockade, or iron metabolism modulation—have demonstrated potential for increasing treatment sensitivity and improving clinical outcomes.²⁸

STRA6 (stimulated by retinoic acid 6) encodes a transmembrane receptor that mediates cellular retinol uptake through its binding to the retinol-retinol-binding protein (RBP) complex, thereby regulating vitamin A metabolism.²⁹ As a central element in vitamin A signaling, STRA6 supports cell growth, differentiation, and metabolic homeostasis, while also modulating key oncogenic pathways, including JAK2/STAT3, mitogen-activated protein kinase (MAPK), and PI3K/Akt.²⁹ Beyond these established roles, STRA6 exhibits oncogenic properties in multiple cancers. Its overexpression enhances proliferation, migration, and invasion and associates with a poor prognosis in colorectal cancer through JAK2/STAT3 activation or metabolic reprogramming.³⁰ In non-small cell lung cancer, however, elevated STRA6 expression correlates with tumor suppression and improved survival,³¹ revealing tissue-specific and context-dependent functions. The role of STRA6 in CNS tumors, especially gliomas, remains poorly defined. Considering the pronounced heterogeneity and metabolic rewiring in gliomas, STRA6 may influence tumor behavior by modulating retinol metabolism, redox equilibrium, or lipid signaling pathways.³² Clarifying the function of STRA6 in glioma could therefore advance our understanding of tumor metabolism and uncover new therapeutic targets and biomarkers.

In this study, we observed elevated STRA6 expression in glioma tissues and cell lines, and its knockdown suppressed glioma cell proliferation, migration, and invasion. Mechanistically, STRA6 deficiency may promote ferroptosis in glioma cells by inhibiting antioxidant stress-related pathways.

Results

STRA6 is highly expressed in glioma tissues and glioma cell lines and is associated with poor prognosis

Bioinformatics analysis of Chinese Glioma Genome Atlas (CGGA: http://www.cgga.org.cn/) and The Cancer Genome Atlas (TCGA: https://portal.gdc.cancer.gov/) data indicated that STRA6 expression was significantly enriched in glioma subtypes with greater malignant potential. The analysis further showed that STRA6 expression correlated with WHO grade, 1p/19q co-deletion, MGMT promoter methylation, and IDH mutation status in glioma (Figure S1A). Kaplan-Meier survival analysis of both datasets revealed that patients with high STRA6 expression had significantly poorer outcomes (Figure S1B). STRA6 was also enriched in the mesenchymal subtype of GBM, the most aggressive form, with an area under the Receiver Operating Characteristic (ROC) curve of 74.4% in CGGA and 76.6% in TCGA, highlighting its subtype specificity (Figure S1C). Western blot (WB) analysis of clinical samples confirmed elevated STRA6 expression in GBM tissues (Figure S1D). Subsequent WB experiments also demonstrated higher STRA6 levels in glioma cell lines (LN229, U87, and U251) compared to normal astrocytes (HA) (Figure S1E). Through immunohistochemical analysis, we found that STRA6 expression was significantly elevated in tumor samples from glioma patients (Figure S2A). Together, these results identify STRA6 as a prognostic biomarker in glioma and suggest its potential relevance for improving patient outcomes.

Downregulation of STRA6 inhibits the growth, migration, and invasion of glioma cells

Stable knockdown of STRA6 was achieved in U251, U87, and GL261 cell lines using lentiviral delivery of shRNA, with WB confirming the reduction in protein expression (Figures 1A–1C). CCK-8 assays revealed that STRA6 downregulation inhibited the proliferation of all three glioma cell lines relative to the control group (Figures 1D–1F). Colony formation assays further corroborated this anti-proliferative effect (Figures 1G–1I). The effects of STRA6 knockdown on glioma cell migration and invasion were comprehensively evaluated through relevant assays. The results showed that in the Transwell assay, the number of STRA6-silenced cells traversing the membrane was markedly reduced (Figures 1J–1L). Similarly, in the wound-healing assay, these cells exhibited a significantly slower rate of scratch closure (Figures 1M–1O). Together, these findings consistently demonstrate that STRA6 knockdown effectively suppresses the migratory capabilities of glioma cells.

Figure 1.

Downregulation of STRA6 inhibits the growth, migration, and invasion of glioma cells

(A–C) Western blotting verified the knockdown efficiency of STRA6 in U251, U87, and GL261 cells.

(D–F) CCK-8 assays were used to assess the effect of STRA6 downregulation on cell proliferation.

(G–I) Colony formation assays further confirmed the inhibitory effect of STRA6 downregulation on cell proliferation.

(J–L) The Transwell assay showed that STRA6 downregulation markedly reduced the migration and invasion of U251, U87, and GL261 cells, with scale bars of 100 μm.

(M–O) The wound-healing assay further confirmed that STRA6 downregulation also suppressed the migratory ability of these cell lines, with scale bars of 200 μm.

For the WB grayscale analysis, Student’s t test was used. n = 3. Statistical analysis of cell proliferation was performed using two-way analysis of variance (ANOVA), n = 6. Data are presented as mean ± standard deviation (SD). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Effects of STRA6 on ferroptosis and the expression of Nrf2, HO1, and GPX4 in glioma cells

To investigate how STRA6 promotes glioma expansion, we compared the transcriptomes of STRA6-silenced and control cells (Figure 2A). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis identified ferroptosis as the most significantly altered pathway (Figure 2B), suggesting a role for STRA6 in redox-regulated cell death. Following STRA6 knockdown, a marked alteration in redox homeostasis was observed in U251, U87, and GL261 cells; intracellular GSH levels were significantly reduced (Figures 2C–2E), indicating weakened antioxidant capacity, while the lipid peroxidation product MDA levels were markedly increased (Figures 2F–2H), reflecting enhanced oxidative stress. These findings suggest that STRA6 downregulation disrupts the redox balance in glioma cells. Ferrous ions (Fe²⁺) constitute a vital component of the intracellular active iron pool, exhibiting potent redox activity. Therefore, we further examined intracellular ferrous ion (Fe²⁺) levels in STRA6 knockdown cells compared to controls. Results revealed that compared to the control group, STRA6 knockdown significantly elevated intracellular Fe²⁺ levels (Figures S2B–S2D), increasing cellular iron load and further enhancing susceptibility to ferroptosis. WB further demonstrated that STRA6 depletion markedly reduced the expression of NRF2, HO1, and GPX4 in U251, U87, and GL261 cells (Figures 2I–2K and S2E–S2G). Based on the above findings, STRA6 may promote the survival of glioma cells by inhibiting ferroptosis through the maintenance of active Nrf2, HO1, and GPX4-related antioxidant pathways. We further assessed the impact of STRA6 knockdown on intracellular ROS levels by flow cytometry. Compared with the control group, U251, U87, and GL261 cells with reduced STRA6 expression consistently exhibited a marked increase in intracellular ROS (Figures 2L–2N), indicating compromised antioxidant defenses and heightened oxidative stress. To exclude interference from other cell death pathways, we further examin-d the expression of apoptosis-related proteins BCL2, BAX, Caspase-3, and Cleaved-Caspase-3. WB analysis revealed no significant changes in the expression levels of these proteins (Figures S3A–S3C), indicating that STRA6 downregulation does not induce cell death by activating classical apoptotic pathways.

Figure 2.

Effects of STRA6 on ferroptosis and the expression of Nrf2, HO1, and GPX4 in glioma cells

(A) RNA-seq analysis of differentially expressed genes between the stable STRA6-knockdown and control groups.

(B) KEGG pathway enrichment showing significant enrichment of the ferroptosis pathway.

(C–E) Downregulation of STRA6 resulted in a marked decrease in intracellular GSH levels.

(F–H) Downregulation of STRA6 also led to an increase in the lipid peroxidation product MDA.

(I–K) Expression of ferroptosis-related genes NRF2, HO1, and GPX4 was markedly decreased in STRA6-knockdown U251, U87, and GL261 cells.

(L–N) Intracellular ROS levels in the U251, U87, and GL261 cell lines.

Student’s t test was used for the statistical analysis of GSH and MDA levels. n = 6. Data are presented as mean ± standard deviation (SD). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Overexpression of STRA6 promotes malignant phenotypes of glioma cells

We established STRA6-overexpressing cell lines via lentiviral transduction in U251, U87, and GL261 GBM models, with expression confirmed by WB (Figures 3A–3C). Overexpression of STRA6 markedly enhanced the proliferative capacity of GBM cells,CCK-8 assays showed a clear increase in cell proliferation, indicating that elevated STRA6 promotes cell viability and growth rate (Figures 3D–3F). Colony formation assays further supported this finding, as STRA6-overexpressing cells formed significantly more and larger colonies, demonstrating enhanced long-term proliferative potential (Figures 3G–3I). The Transwell assay showed that the migratory ability of cells overexpressing STRA6 was significantly enhanced (Figures 3J–3L). The wound-healing assay demonstrated that STRA6-overexpressing cells closed the scratch area much faster, indicating an increased migration efficiency (Figures 3M–3O). From a biochemical perspective, overexpression of STRA6 significantly increased intracellular GSH levels (Figures 3P–3R). At the same time, STRA6 overexpression markedly reduced the levels of the lipid peroxidation product MDA (Figures 3S–3U). WB analysis also indicated upregulation of the ferroptosis-related proteins NRF2, HO1, and GPX4 in glioma cells (Figures 3V–3X and S3D–S3F). Together, these findings demonstrate that STRA6 overexpression promotes malignant progression in glioma by reinforcing antioxidant defenses and suppressing ferroptosis.

Figure 3.

Overexpression of STRA6 promotes malignant phenotypes of glioma cells

(A–C) Stable STRA6-overexpressing U251, U87, and GL261 cell lines.

(D–F) CCK-8 assay results showed a significant increase in cell proliferation.

(G–I) Colony formation assays further confirmed that the colony-forming ability was also markedly enhanced.

(J–L) The Transwell assay showed that the migratory ability of cells overexpressing STRA6 was significantly enhanced, with scale bars of 100 μm.

(M–O) The wound-healing assay indicated that STRA6-overexpressing cells exhibited increased migration efficiency, with scale bars of 200 μm.

(P–R) From a biochemical perspective, overexpression of STRA6 significantly increased intracellular GSH levels.

(S–U) STRA6 overexpression markedly reduced the levels of the lipid peroxidation product MDA.

(V–X) Expression levels of ferroptosis-related proteins NRF2, HO1, and GPX4 were increased in STRA6-overexpressing U251, U87, and GL261 cells.

Statistical analysis of cell proliferation was performed using two-way analysis of variance (ANOVA) n = 6. Student’s t test was used for the statistical analysis of GSH and MDA levels. n = 6. Data are presented as mean ± standard deviation (SD). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

STRA6 influences ferroptosis in GBM cells by regulating NRF2

NRF2, a key transcription factor regulating antioxidant responses and influencing ferroptosis susceptibility, relies on nuclear translocation for its function. We examined the intracellular distribution of NRF2 via nuclear-cytoplasmic separation experiments. Results showed that STRA6 knockdown markedly reduced NRF2 nuclear translocation (Figures 4A–4C), suggesting suppressed transcriptional activity and potential impairment of NRF2-mediated antioxidant and cell protective functions. Collectively, these findings indicate that STRA6 may participate in cellular stress responses by regulating NRF2 nuclear translocation status, thereby influencing ferroptosis susceptibility. To further investigate the role of NRF2, we administered the NRF2 agonist tert-Butylhydroquinone (TBHQ) to the STRA6 knockdown model. Results showed that compared to untreated knockdown controls, TBHQ treatment led to a partial recovery of NRF2 protein expression levels (Figures 4D–4F and S3G–S3I). This finding indicates that TBHQ enhances NRF2 pathway activity in the context of STRA6 knockdown, providing experimental evidence for subsequent studies elucidating the STRA6-NRF2 axis’s involvement in regulating oxidative stress and ferroptosis.

Figure 4.

STRA6 influences ferroptosis in glioblastoma cells by regulating NRF2

(A–C) Nuclear localization of NRF2 protein following STRA6 knockdown.

(D–F) Expression levels of NRF2 protein following TBHQ treatment.

For the WB grayscale analysis, Student’s t test was used. n = 3. Data are presented as mean ± standard deviation (SD). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Ferroptosis inhibitors rescued the antitumor effect induced by STRA6 downregulation

Ferroptosis inhibitors are a class of compounds that reduce ferroptosis by inhibiting iron-dependent lipid peroxidation. Ferrostatin-1, a ferroptosis inhibitor,³³ significantly restored the proliferative capacity of STRA6-knockdown U251 and U87 cells (Figures 5A and 5B). Colony formation assays showed that Ferrostatin-1 partially reversed the decrease in clonogenic capacity caused by STRA6 knockdown (Figures 5C and 5D). Transwell assays demonstrated that Ferrostatin-1 treatment could partially restore the migratory ability of STRA6-knockdown cells (Figures 4E and 4F). Wound-healing assays further confirmed this result, as Ferrostatin-1 treatment markedly accelerated the scratch closure of STRA6-knockdown cells, indicating a partial recovery of their migratory capacity (Figures 5G and 5H). Furthermore, intracellular measurements indicated that Ferrostatin-1 elevated GSH levels and reduced MDA levels in STRA6-deficient cells (Figures 5I–5L). WB analysis also confirmed that Ferrostatin-1 partially restored the expression of the antioxidant proteins NRF2, HO1, and GPX4 (Figures 5M, 5N, S4A, and S4B). Collectively, these findings indicate that pharmacological inhibition of ferroptosis is sufficient to counteract the cytostatic and anti-migratory effects induced by STRA6 loss. To complement and cross-validate the findings from Ferrostatin-1, we further employed the iron chelator Deferoxamine (DFO) as an intervention. Results demonstrated that adding DFO to STRA6-downregulated cells led to varying degrees of recovery in the expression of key antioxidant/anti-lipid peroxidation-related proteins, such as NRF2, HO1, and GPX4 compared to untreated knockdown groups (Figures S4C and S4D).

Figure 5.

Ferroptosis inhibitors rescued the antitumor effect induced by STRA6 downregulation

(A and B) Proliferation ability of STRA6-knockdown cells after Ferrostatin-1 treatment.

(C–H) Results of colony formation, Transwell, and wound healing assays.

The scale bars represent 100 and 200 μm, respectively.

(I–L) Intracellular GSH and MDA levels after treatment.

(M and N) Expression of ferroptosis-related proteins NRF2, HO1, and GPX4 in U251 and U87 cells with stable STRA6 knockdown following Ferrostatin-1 treatment.

Statistical analysis of cell proliferation was performed using two-way analysis of variance (ANOVA), n = 6. Student’s t test was used for the statistical analysis of GSH and MDA levels. n = 6. Data are presented as mean ± standard deviation (SD). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

The tumor-promoting effect of STRA6 overexpression can be inhibited by Erastin and the RBP4 antagonist A1120

Following STRA6 overexpression, the cells developed a pronounced resistance to ferroptosis. To confirm this STRA6-mediated suppression of ferroptosis, we treated the cells with the ferroptosis inducer Erastin. Erastin treatment significantly inhibited the proliferation of STRA6-overexpressing cells (Figures 6A and 6B), impaired colony formation (Figures 6C and 6D), and suppressed their migratory capacities (Figures 6E and 6F), while also delaying wound closure (Figures 6G and 6H). These results indicate that Erastin partially reversed the ferroptosis suppression caused by STRA6 overexpression. Further analysis showed that STRA6 overexpression elevated intracellular GSH levels and reduced MDA levels, whereas Erastin treatment decreased GSH and increased MDA (Figures 6I–6L), further confirming that Erastin counteracts the ferroptosis inhibition mediated by STRA6. Meanwhile, Erastin treatment also reduced the expression levels of the ferroptosis-related proteins NRF2, HO1, and GPX4 (Figures 6M, 6N, S4E, and S4F). In summary, Erastin counteracts the STRA6-overexpression-induced promotion of glioma cell proliferation, migration, and invasion. In glioma cells, RBP4-STRA6 constitutes the core axis mediating retinol delivery and cellular uptake. To investigate whether retinol metabolism participates in regulating ferroptosis susceptibility in glioma cells, we treated cells with the RBP4 antagonist A1120 to block RBP4-mediated retinol delivery. Results showed that under STRA6 overexpression, A1120 treatment led to a decrease in the expression levels of ferroptosis-related proteins NRF2, HO1, and GPX4 (Figures S5A and S5B).

Figure 6.

The tumor-promoting effect of STRA6 overexpression can be inhibited by Erastin and the RBP4 antagonist A1120

(A and B) Proliferation ability of STRA6-overexpressing cells after Erastin treatment.

(C and D) Results of colony formation.

(E and F) Results of Transwell, with scale bars of 100 μm.

(G and H) Results of wound healing assays, with scale bars of 200 μm.

(I–L) Intracellular GSH and MDA levels after treatment.

(M and N) Expression of ferroptosis-related proteins NRF2, HO1, and GPX4 in U251 and U87 cells with stable STRA6 overexpression following Erastin treatment.

Statistical analysis of cell proliferation was performed using two-way analysis of variance (ANOVA) n = 6. Student’s t test was used for the statistical analysis of GSH and MDA levels. n = 6. Data are presented as mean ± standard deviation (SD). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

STRA6 promotes the in vivo growth of GBM cells

To investigate the role of STRA6 in GBM progression in vivo, we established STRA6-overexpressing or STRA6-knockdown GBM cells and subcutaneously injected them into C57BL/6J mice to generate allograft tumors. The results showed that STRA6 knockdown suppressed tumor growth (Figure 7A), reduced tumor weight (Figure 7B), and decreased tumor size (Figures 7C and 7D). In contrast, the STRA6-overexpressing group exhibited the opposite effects compared with the control group (Figures 7E–7H). Concurrently, body-weight changes were recorded for both experimental groups. The results show that mice in the STRA6 knockdown group exhibited higher body weights than the control group, while STRA6 overexpression produced the opposite effect (Figures 7I and 7J). WB analysis was performed to examine the expression of ferroptosis-related signaling proteins in allograft tumors with STRA6 knockdown or overexpression. The results revealed decreased protein levels of NRF2, HO1, and GPX4 in STRA6-knockdown tumors, whereas these proteins were upregulated in STRA6-overexpressing tumors (Figures 5C, 5D, 7K, and 7L). At the same time, we examined changes in additional ferroptosis-related proteins (Figures S5E and S5F). Based on the abovementioned findings, STRA6 may promote the growth of GBM cells in vivo by regulating pathways associated with NRF2, HO1, and GPX4.

Figure 7.

STRA6 promotes the in vivo growth of GBM cells

(A) Photographs show subcutaneous transplanted tumors formed by cells with STRA6 knockdown and controls through subcutaneous injection.

(B) Tumor weight of the two groups.

(C and D) Tumor volume was assessed at 4-day intervals after subcutaneous injection, with measurements recorded on the day tumors were harvested from the specified group.

(E) Photographs show subcutaneous transplanted tumors formed by cells with STRA6 overexpression and controls through subcutaneous injection.

(F) Tumor weight of the two groups.

(G and H) Tumor volume was assessed at 4-day intervals after subcutaneous injection, with measurements recorded on the day tumors were harvested from the specified group.

(I and J) Body weight changes of the mice.

(K–L) Changes in NRF2, HO1, and GPX4 in tumors with STRA6 knockdown and overexpression. Measure the longest diameter (L) and shortest diameter (W) of the tumor using a Vernier caliper, and calculate the tumor volume using the formula V = (L × W²)/2. Simultaneously, determine the tumor mass. Monitor and record changes in mouse body weight and tumor volume at regular intervals; intergroup comparisons were performed using Student’s t test. n = 5.

Data are presented as mean ± standard deviation (SD). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Discussion

STRA6 localizes to the cell membrane and cytoplasm, where it functions as a key transporter for maintaining intracellular retinol homeostasis.³⁴ Its overexpression in multiple cancers is closely associated with tumorigenesis, progression, and patient prognosis.^35,36,37 However, the function and potential mechanisms of STRA6 in GBM, remain unclear. Our findings demonstrate that STRA6 is highly expressed in gliomas and exhibits potential biological functions as a prognostic biomarker for glioma patients.

In this study, we found that STRA6 is highly expressed in GBM tissues and various GBM cell lines. Further clinical data analysis showed that the high expression of STRA6 is closely related to larger tumor volume, higher malignancy, and poorer histological type. Functional experiments indicated that STRA6 significantly promotes the proliferation, migration, and invasion abilities of GBM cells, suggesting its oncogenic role in tumor progression. These results suggest that STRA6 is not only upregulated in GBM but may also participate in malignant progression by regulating cell proliferation- and invasion-related pathways. Therefore, STRA6 holds promise as a potential therapeutic target for GBM, offering new strategies for future molecular-targeted therapies. Further mechanistic studies will help reveal the specific pathways involved in STRA6’s role in GBM and promote its clinical diagnostic and therapeutic applications.

Ferroptosis, a novel form of programmed cell death distinct from apoptosis, necrosis, and autophagy, is driven by the excessive accumulation of intracellular iron ions and lipid peroxidation reactions.²⁰ Lipid peroxidation plays a crucial role in the occurrence and development of ferroptosis, primarily manifested by the accumulation of ROS, which leads to the oxidative degradation of PUFAs in the cell membrane. This, in turn, triggers the destruction of cell membrane structure and loss of function.³⁸ In recent years, ferroptosis and its potential role in gliomas have become a hot topic in cancer research.³⁹ Regulating ferroptosis is considered a potential strategy for the treatment of gliomas. This study aims to explore the role and mechanism of STRA6 in the ferroptosis process of glioma cells. The results indicate that downregulation of STRA6 significantly promotes the occurrence of ferroptosis, while high expression of STRA6 significantly inhibits this process. This finding suggests that STRA6 may influence glioma growth and invasion by regulating ferroptosis, offering a new potential therapeutic target for glioma treatment.

The NRF2/HO1 axis is a critical antioxidant signaling pathway that helps cells respond to oxidative stress. It plays a central role in maintaining cellular homeostasis, regulating iron homeostasis, and controlling the type of cell death.⁴⁰ NRF2, as a key transcription factor, is activated when cells are exposed to oxidative stress or stimuli such as ionizing radiation. Upon activation, NRF2 translocates to the cell nucleus, where it binds to the antioxidant response elements (AREs) and induces the expression of a series of antioxidant genes, including HO1.⁴¹ HO1, as an important downstream effector molecule, exerts antioxidant, anti-inflammatory, and cell-protective effects by degrading heme to produce carbon monoxide, free iron, and biliverdin. This process effectively reduces the accumulation of ROS and alleviates oxidative stress-induced damage to cells.⁴² The NRF2/HO1 axis plays a significant role in regulating ferroptosis. The NRF2/HO1 axis enhances the cell’s antioxidant capacity, inhibits ROS production, and reduces lipid peroxidation levels, thereby blocking the occurrence of ferroptosis to some extent.⁴³ Additionally, the products released by HO1 play an important role in regulating cellular iron homeostasis, inhibiting inflammatory responses, and protecting cell membrane integrity, further strengthening its function in ferroptosis regulation.⁴⁴ Therefore, an in-depth investigation of the regulatory mechanisms of the NRF2/HO1 axis not only helps elucidate cellular biological responses under stress conditions but also provides a crucial theoretical foundation and practical guidance for developing novel therapeutic strategies targeting ferroptosis and related diseases.

In this study, we analyzed the expression changes of NRF2, HO1, and GPX4 following the downregulation and upregulation of STRA6 through in vitro cell experiments and in vivo animal models. Results indicated that high STRA6 expression significantly suppresses ferroptosis in GBM cells while enhancing activation of the NRF2/HO1/antioxidant axis. This suggests STRA6 may block ferroptosis by activating this antioxidant pathway, reducing intracellular ROS levels, and inhibiting lipid peroxidation. These findings not only reveal STRA6’s pivotal role in regulating ferroptosis in glioma but also provide novel theoretical support for developing STRA6-targeted therapeutic strategies against glioma. Future investigations into the specific molecular mechanisms by which STRA6 modulates ferroptosis will advance its clinical translation for precision therapy in glioma.

Limitations of the study

Although this study has made some progress, several limitations remain. First, the specific mechanisms underlying the interactions between STRA6 and the NRF2/GPX4 and ferroptosis-related pathways have not yet been fully elucidated, and their molecular regulatory relationships require further in-depth investigation. Second, the precise biological roles of standard retinol and RBP4, as well as the exact effects of STRA6-mediated alterations in retinol metabolism on the ferroptosis process, remain unclear and require further exploration through metabolomics, molecular biology, and functional experiments. Additionally, the in vivo experiments in this study were conducted using a subcutaneous tumor model; validation using an in situ intracranial GBM model, which more closely mimics clinical reality, has not yet been performed. Further studies in this regard will be conducted in the future.

Resource availability

Lead contact

Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Lanlan Zhang (zll3526029@163.com).

Materials availability

This study did not generate new unique reagents.

Data and code availability

• •All data reported in this paper will be shared by the lead contact upon request.
• •This paper does not report original code.
• •Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

This work was supported by the Scientific Research Foundation of Education Department of Anhui Province (grant nos. 2024AH040093 and 2025AHGXZK50153), Chuzhou Science and Technology Program (grant no. 2024YF007), and the Health Research Program of Chuzhou (grant no. CZWJ2024A001).

Author contributions

T.Y., C.Z., D.T., Z.Y., Z.W., and J.W. performed the experiments and collected the data. T.Y., C.Z., and D.T. analyzed the data and prepared the figures. T.Y. and C.Z. wrote the manuscript. N.L., H.Z., and L.Z. supervised the study and revised the manuscript. All authors approved the final version.

Declaration of interests

The authors declare no conflict of interests.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work, the authors used ChatGPT for language polishing to improve the readability of the manuscript. After using this tool/service, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-Actin	Proteintech	RRID:AB_2883475
Anti-GPX4	Proteintech	RRID:AB_3747236
Anti-NRF2	Proteintech	RRID:AB_2919830
Anti-HO1	ZENBIO	RRID:AB_3083512
Anti-Flag	Sigma-Aldrich	RRID:AB_262044
Anti-BCL2	Abcam	RRID:AB_725644
Anti-BAX	Abcam	RRID:AB_725631
Anti-Caspase-3	Proteintech	RRID:AB_10733244
Anti-Cleaved-Caspase-3	Proteintech	RRID:AB_3073913
Anti-STRA6	ImmunoWay	RRID:AB_3752062
Anti-Lamin B1	Proteintech	RRID:AB_2136290
Anti-ACSL4	Proteintech	RRID:AB_2832995
Anti-XCT	Affinity	RRID:AB_2845314
Anti-FTH1	Abcam	RRID:AB_1267146
Bacterial and virus strains
DH5α	Weidibio	DL1001S
Biological samples
Human tissue	The First People’s Hospital of Chuzhou	Ethics Number:2024-47
Animal tissue	Anhui Medical University	Ethics Number:LLSC20221212
Chemicals, peptides, and recombinant proteins
Ferrostatin-1	MCE	HY-100579
Erastin	MCE	HY-15763
TBHQ	MCE	HY-100489
A1120	MCE	HY-107633
Deferoxamine	MCE	HY-B1625
Puromycin	Biosharp	BL528B
Invitrogen™ Lipofectamine™ 2000	Invitrogen	11668019
Critical commercial assays
	Biosharp	N/A
Cell Counting Kit-8	Goonie	100-120-1000
Cell Malondialdehyde (MDA) assay kit	Nanjing Jiancheng Bioengineering Institute	A003-4-1
Reduced glutathione (GSH) assay kit	Nanjing Jiancheng Bioengineering Institute	A006-2-1
Ferric and Ferrous Ion Assay Kit	Beyotime	S1066S
Nuclear and Cytoplasmic Protein Extraction Kit	Beyotime	P0027
Reactive Oxygen Species Assay Kit	Beyotime	S0033S
Deposited data
RNA-seq	Beijing Genomics Institute	NCBI SRA: PRJNA1373143
Experimental models: Cell lines
U251	ProCell Life Science & Technology	CM-0237
U87 MG	ProCell Life Science & Technology	CL-0238
LN229	ProCell Life Science & Technology	CL-0578
293T	ProCell Life Science & Technology	CL-0469
GL261	IMMOCELL	IM-M084
HA1800	IMMOCELL	IM-H437777
Experimental models: Organisms/strains
Mouse: C57BL/6J	GemPharmatech LLC	RRID:IMSR_JAX:000664
Oligonucleotides
shSTRA6#1	Wuhan Miaoling Biotechnology	5′-GCTACTACACGTACCGAAACT-3′
shSTRA6#2	Wuhan Miaoling Biotechnology	5′-GCTCTGGAAGTGTGCTACA-3′
Recombinant DNA
psPAX2	Wuhan Miaoling Biotechnology	RRID:Addgene_12260
pMD2.G	Wuhan Miaoling Biotechnology	RRID:Addgene_12259
Software and algorithms
ImageJ	Schindelin et al.	RRID:SCR_003070
Adobe Photoshop	Adobe Inc.	RRID:SCR_014199
GraphPad Prism	GraphPad Software	RRID:SCR_002798
Flowjo	BD Biosciences	RRID:SCR_008520

Experimental model and study participant details

All animal experiments were approved by the Institutional Animal Care and Use Committee of Anhui Medical University (Animal Ethics Number: LLSC20221212). The mouse model used in this study was C57BL/6J mice (4 weeks old, male). Mice were maintained under specific pathogen-free conditions at 22 ± 2°C with a 12-h light/dark cycle and had free access to food and water. Only male mice were used in this study to minimize hormonal variation. Therefore, potential sex-specific differences were not evaluated, which should be considered a limitation of the study. Three paired GBM tissues and adjacent brain tissues were obtained from patients undergoing surgical resection at the Department of Neurosurgery, The First People’s Hospital of Chuzhou, grade IV (n = 3). Written informed consent was obtained from all patients or their legal guardians. The study was approved by the Institutional Ethics Committee of The First People’s Hospital of Chuzhou (Approval No. 2024-47). Both male and female patients were included. However, sex-based analyses were not performed because of the limited sample size, which represents a limitation of the present study. The human glioblastoma cell lines U251, U87 MG, LN229, and the human embryonic kidney cell line 293T were purchased from ProCell Life Science & Technology Co., Ltd. (Wuhan, China). The human astrocyte cell line HA1800 and the murine glioma cell line GL261 were obtained from IMMOCELL Biotechnology Co., Ltd. (Xiamen, China). All cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) under standard conditions at 37°C in a humidified incubator with 5% CO₂.

Method details

Bioinformatics analyses

Bioinformatics analyses utilized data from multiple public databases. Transcriptomic and clinical data for GBM and normal tissues were sourced from The Cancer Genome Atlas (TCGA, https://portal.gdc.cancer.gov/) to evaluate STRA6 expression and its prognostic significance. An independent glioma cohort from The Chinese Glioma Genome Atlas (CGGA, http://www.cgga.org.cn/) served as a validation set. We examined differential STRA6 expression and its correlation with overall survival across tumor types. All data were used in accordance with the open-access policies of the respective databases.

Clinical samples and cell culture

Tissue specimens were collected from patients treated in the Department of Neurosurgery at Chuzhou First People’s Hospital, following informed consent and with approval from the Ethics Committee. The human GBM cell lines U251 and U87MG, along with the LN229 cell line, were sourced from ProCell Life Science & Technology Co., Ltd. (Wuhan, China). HA1800 and GL261 cell lines were purchased from IMMOCELL (Xiamen, China). Cells were maintained at 37°C in a 5% CO2 atmosphere using high-glucose DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, with the medium replaced regularly. Prior to experimental use, all cell lines were verified to be free of mycoplasma contamination.

Construction of stable GBM cell lines

Stable cell lines with either STRA6 overexpression or knockdown were generated via lentiviral transduction. Plasmids encoding STRA6 overexpression or shRNA constructs were co-transfected with the packaging plasmids pMD2.G and psPAX2 into HEK293T cells using Lipofectamine 2000 (Invitrogen, USA). Viral supernatants were harvested at 36 and 72 h, filtered, and concentrated with PEG8000 at 4°C. These lentiviral preparations were then used to infect GBM cells (U87MG, U251, or GL261). After 48 h, cells were selected with 10 μM puromycin to establish stable cell lines, and STRA6 expression levels were confirmed by Western blotting. The final concentration of DMSO was 1 μM. shSTRA6#1:5′-GCTACTACACGTACCGAAACT-3′, shSTRA6#2:5′-GCTCTGGAAGTGTGCTACA-3’.

Cell proliferation assay

Cell proliferation was assessed with a CCK-8 kit (GOONIE, China). Cells in the logarithmic growth phase were plated in 96-well plates at a density of 2 × 10³ cells per well and maintained at 37°C with 5% CO2. Following a 2-h attachment period, CCK-8 solution was added for the initial (0-h) measurement, with subsequent measurements taken at 24, 48,72 and 96 h. After each 2-h incubation with the reagent, the absorbance at 450 nm was measured using a microplate reader to determine cell proliferation.

Settlement formation experiment

For the colony formation assay, cells were seeded into 6-well plates at a density of 1 × 10³ cells per well and cultured under standard conditions (37°C, 5% CO2) for 8–12 days. Once visible colonies had formed, the cells were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet. After washing and drying, the colonies were evaluated based on their number and size.

Wound healing assays

For the wound healing assay, U251 and U87 cells were seeded into 6-well plates and cultured to approximately 80% confluence. A straight scratch was then introduced into the cell monolayer with a sterile pipette tip, and any dislodged cells were washed away with PBS. The medium was subsequently replaced with DMEM containing 1% FBS. Images of identical wound regions were taken at 0, 24, and 48 h, and the wound width and healing area were measured with ImageJ software to assess cell migration.

Transwell migration assays

Cell migration assays were conducted in Transwell chambers. We resuspended 2 × 105 cells in 200 μL of serum-free DMEM and placed them in the upper chamber, while the lower chamber received 800 μL of DMEM supplemented with 20% FBS as a chemoattractant. Following a 24-h incubation at 37°C with 5% CO2, non-migrated cells on the upper membrane surface were carefully removed. Migrated cells were fixed with 4% paraformaldehyde and stained using 0.1% crystal violet. These stained cells were then examined under a microscope, and randomly selected fields were photographed to enable cell counting and statistical analysis.

Western blotting

Total cellular and tissue proteins were extracted with RIPA lysis buffer (Beyotime, China) supplemented with protease and phosphatase inhibitors. Following lysis on ice, the samples were centrifuged at 12,000× g for 15 min at 4°C, and the supernatants were collected. Protein concentrations were determined using a BCA assay, and equal amounts of protein were denatured at 95°C for 5 min, resolved by SDS-PAGE, and subjected to Western blot analysis. The primary antibodies used were anti-Actin, anti-GPX4, and anti-NRF2 (Proteintech, China); anti-HO1 (ZENBIO, China); anti-Flag (Sigma-Aldrich, USA); and anti-STRA6 (ImmunoWay, China).

Detection of MDA and GSH levels

Intracellular MDA and GSH levels were quantified with commercial assay kits (Nanjing Jiancheng, China). Following attachment in 6-well plates, U251 and U87MG cells were processed as specified by the manufacturer. The resulting MDA and GSH measurements served to assess the cellular oxidative stress status.

Detection of ferrous iron levels in cells

Intracellular ferrous ion (Fe²⁺) levels were quantified using the Ferric and Ferrous Ion Assay Kit, Colorimetric (Beyotime, China). Procedures were performed according to the manufacturer’s instructions.

Flow cytometry analysis

Flow cytometry was used to detect the effect of STRA6 downregulation on intracellular reactive oxygen species (ROS) levels. ROS detection was performed using a ROS detection kit (Beyotime, China) according to the kit instructions.

Nuclear extraction experiment

Nuclear and cytoplasmic protein extraction was performed using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, China) to detect the respective protein levels in the cell nucleus and cytoplasm.

RNA-sequencing and data analysis

Total RNA was isolated from both STRA6-knockdown and control cells with TRIzol reagent (Invitrogen, USA). These RNA samples were sent to the Beijing Genomics Institute (BGI, China) for sequencing. The resulting data were processed and normalized to enable differential gene expression analysis. The generated RNA-seq datasets have been submitted to the National Center for Biotechnology Information (NCBI) repository under the SRA accession designation (SRA: PRJNA1373143).

Agonists and antagonists

Ferrostatin-1 (MCE, China) were dissolved in dimethyl sulfoxide (DMSO) to prepare 10 mM stock solutions. For treatment, the compounds were diluted to a final concentration of 2 μM with a final DMSO concentration of 0.02%. Deferoxamine (MCE, China) were dissolved in dimethyl sulfoxide (DMSO) to prepare 10 mM stock solutions. For treatment, the compounds were diluted to a final concentration of 5 μM with a final DMSO concentration of 0.05%. Erastin (MCE, China) were dissolved in dimethyl sulfoxide (DMSO) to prepare 10 mM stock solutions. For treatment, the compounds were diluted to a final concentration of 5 μM with a final DMSO concentration of 0.05%. A1120 (MCE, China) were dissolved in dimethyl sulfoxide (DMSO) to prepare 10 mM stock solutions. For treatment, the compounds were diluted to a final concentration of 0.5 μM with a final DMSO concentration of 0.005%. TBHQ (MCE, China) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution. For treatment, the compound was diluted to a final concentration of 5 μM, with a final DMSO concentration of 0.05%. Cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 24 h, after which the relevant parameters were assessed.

Immunohistochemistry

Immunohistochemistry (IHC) was employed to detect and evaluate STRA6 expression in tissues. After completing dewaxing, rehydration, and antigen retrieval of the sections, they were first treated in blocking solution for 30 min to inhibit endogenous peroxidase activity. Subsequently, goat serum was applied for 30 min to reduce non-specific binding. Following washing, the corresponding secondary antibody was added and incubated at room temperature for 30 min. Finally, DAB staining was performed, and images were captured and analyzed under a microscope.

Animal models

To investigate the role of STRA6 in the progression of glioblastoma, GBM cell lines stably expressing either overexpressed or knockdown versions of STRA6 were established. Cells in the logarithmic growth phase were digested, counted, and resuspended in sterile PBS. They were then mixed with an equal volume of matrix gel at a concentration of 3 × 10⁶ cells/100 μL and subcutaneously injected into C57BL/6J mice to establish allograft tumor models (5 mice per group). Tumor growth was observed and recorded every 4 days post-injection. The longest diameter (L) and shortest diameter (W) of the tumors were measured using a Vernier caliper, and tumor volume was calculated using the formula V = (L × W²)/2 to plot tumor growth curves. Concurrently, changes in mouse body weight were monitored and recorded periodically to assess tumor progression and the animals’ general condition. At the end of the experiment, mice were euthanized, tumor tissues were excised and weighed, and subsequent molecular and histological analyses were performed. Statistical analysis was conducted using GraphPad Prism software, and data are expressed as mean ± standard deviation (mean ± SD). Comparisons between groups were performed using Student’s t test. A p-value <0.05 was considered statistically significant.

Statistical analysis

Data analysis was conducted using GraphPad Prism software. All results are presented as mean ± standard deviation (SD). Differences between groups were assessed by Student’s t test or two-way ANOVA. A p-value below 0.05 was considered statistically significant.

Quantification and statistical analysis

All images and data were processed using Adobe Photoshop, ImageJ, GraphPad Prism, and FlowJo software.

Image processing and quantification

Western blot (WB) grayscale analysis in all figures was performed using Student’s t test (n = 3). Cell proliferation data in Figures 1, 3, 5, and 6 were analyzed using two-way analysis of variance (ANOVA) (n = 6). GSH and MDA levels in Figures 2, 3, 5, and 6 were analyzed using Student’s t test (n = 6). For the tumor xenograft assay (Figure 7), the longest diameter (L) and shortest diameter (W) of each tumor were measured using a Vernier caliper, and tumor volume was calculated using the formula V = (L × W²)/2. Tumor weight was also determined. Mouse body weight and tumor volume were monitored at regular intervals, and comparisons between groups were performed using Student’s t test (n = 5). Iron ion content in Figure S2 was analyzed using Student’s t test (n = 3). Data are presented as mean ± standard deviation (SD). Statistical significance was defined as p < 0.05, p < 0.01, and p < 0.001.