WT1-AS acts as a tumor suppressor in cervical cancer via OSR2-mediated transcriptional activation

Introduction

Cervical cancer (CESC) continues to be a major public health issue worldwide (1,2), particularly in regions where access to human papillomavirus (HPV) vaccination and routine screening is limited (3,4). Although advances in prevention and early detection have reduced incidence in some populations, a substantial number of patients are still diagnosed at advanced stages, facing poor therapeutic outcomes (5-7). This underscores the urgent need to elucidate molecular events driving CESC development and to identify novel biomarkers that may improve prognosis prediction and therapeutic strategies.

Long non-coding RNAs (lncRNAs), defined as transcripts longer than 200 nucleotides without protein-coding capacity, have emerged as essential modulators of gene regulation (8-10). They are increasingly recognized for their ability to influence chromatin remodeling (11,12), transcriptional activity (13), and post-transcriptional modifications (14). Aberrant lncRNA expression has been linked to cancer initiation and progression, with documented roles in processes such as proliferation, apoptosis, invasion, and immune regulation (15-17). In CESC, several lncRNAs, such as HOTAIR and MALAT1, have been reported to contribute to tumor progression and aggressive phenotypes (18,19), indicating that lncRNA-mediated regulatory networks are deeply involved in disease development. These observations support the view that the identification of functionally relevant lncRNAs may provide new insight into the molecular heterogeneity of CESC.

Wilms tumor 1 antisense RNA (WT1-AS), an antisense transcript located opposite to the WT1 gene (20), has drawn attention in recent years due to its involvement in different cancers (21-24). Previous studies suggest that WT1-AS may function as a tumor suppressor by modulating oncogenic pathways, reduced WT1-AS expression has been associated with enhanced proliferation and invasion in gastric cancer (26), while in triple-negative breast cancer WT1-AS inhibits migration and invasion by modulating transforming growth factor-β1 signaling (27). In hepatocellular carcinoma, WT1-AS has been shown to promote apoptosis through downregulation of WT1 (28), yet its biological significance and regulatory mechanisms in CESC remain largely unexplored, previous studies have shown that WT1-AS overexpression inhibits CESC progression, suppresses the proliferation of cervical squamous carcinoma cells through upregulation of p53 (29), and attenuates the aggressiveness of CESC cells by modulating the miR-330-5p/p53 axis (30). However, the current evidence remains limited in scope and is largely focused on downstream functional effects and ceRNA-related mechanisms. The broader expression pattern of WT1-AS in CESC, its prognostic significance, its distribution at the single-cell level, and its role in apoptosis have not yet been systematically characterized. In addition, whether WT1-AS is subject to specific upstream transcriptional regulation in CESC remains unclear. Addressing these issues may provide a more integrated understanding of the biological significance and regulatory basis of WT1-AS in CESC.

Among the multiple layers of lncRNA regulation, transcriptional control is particularly important because it determines when and where a lncRNA is expressed and thereby shapes its downstream biological effects. Aberrant transcription factor activity is a hallmark of cancer and may critically influence lncRNA-centered regulatory networks. Thus, clarifying the transcriptional regulation of WT1-AS could help explain its dysregulation and functional impact in CESC. Odd-skipped related transcription factor 2 (OSR2) is a zinc finger transcription factor that has been studied primarily in developmental processes, including craniofacial and organ development (31). Emerging evidence also suggests that OSR2 may have biological relevance in the tumor setting (32-34), although its role in CESC and its potential relationship with lncRNA regulation remain largely unclear. These features make OSR2 a plausible candidate for investigating the upstream transcriptional regulation of WT1-AS.

To address this gap, we conducted a multi-layered investigation of WT1-AS in CESC. Using pan-cancer and The Cancer Genome Atlas (TCGA)-CESC data, we first evaluated its expression and prognostic impact. Functional enrichment and single-cell transcriptome analyses were then applied to predict its biological roles. In addition, in vitro and in vivo experiments were performed to validate its influence on apoptosis and tumor progression. Finally, we identified OSR2 as a transcription factor that directly regulates WT1-AS, establishing a novel OSR2/WT1-AS axis with functional relevance in CESC. The overall workflow of the study is illustrated in Figure 1. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2842/rc).

Figure 1 The overall workflow of the study. This diagram was generated using Nano Banana software based on the author’s own experimental results and data. All graphic elements are original and created entirely by the author; no third-party templates, icons or copyrighted material have been used. CESC, cervical cancer; FIMO; GO, Gene Ontology; JASPAR; KD; KEGG, Kyoto Encyclopedia of Genes and Genomes; lncRNA, long non-coding RNA; OV, ovarian cancer; TCGA, The Cancer Genome Atlas; TNF.

Methods

Data collection and preprocessing

Transcriptomic datasets and clinical annotations from 33 tumor entities were obtained through the UCSC Xena browser (https://xena.ucsc.edu/) and the Genomic Data Commons (https://portal.gdc.cancer.gov/). For the TCGA-CESC cohort, patients were included if WT1-AS expression data and corresponding clinical follow-up information were available. Samples without RNA expression data or overall survival (OS) information were excluded from prognostic analyses. For clinicopathological correlation analyses, cases with missing information for a given variable were excluded from the corresponding subgroup comparison. The clinical parameters analyzed in CESC included age, pathological T stage, lymph node status (N stage), distant metastasis status (M stage), overall pathological stage, and survival status. Differences in WT1-AS transcript levels between malignant and adjacent normal tissues were evaluated using the Wilcoxon rank-sum test.

Survival analysis

Clinical outcome data for patients across TCGA cohorts were used to assess the prognostic impact of WT1-AS. OS served as the main endpoint. Patients were divided into high- and low-expression subgroups based on the median normalized WT1-AS expression value. Kaplan-Meier survival curves were generated with the R packages “survival” and “survminer”, and log-rank tests were applied to compare groups. Hazard ratios were estimated by univariate Cox regression.

Functional enrichment analysis

Genes correlated with WT1-AS expression were analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment through the clusterProfiler package. The Benjamini-Hochberg method was applied for multiple testing correction, and an adjusted P<0.05 was considered statistically significant.

Identification of downstream candidates

Candidate genes potentially regulated by WT1-AS were identified by integrating FIMO motif analysis from the JASPAR database with co-expression analysis in the TCGA-CESC dataset. Genes with significant correlation (|R|>0.25, P<0.05) and prognostic value were retained as potential downstream targets.

Single-cell transcriptome analysis

To explore the cellular distribution of WT1-AS, single-cell RNA-seq data (GSE168652) were downloaded from the TISCH database (http://tisch.comp-genomics.org/). Expression patterns of WT1-AS were visualized across annotated cell subtypes within CESC tissues.

Cell culture and transfection

HeLa and SiHa CESC cell lines were purchased from Zhejiang Mason Cell Technology Co. (Hangzhou, China). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 ℃ with 5% CO₂. WT1-AS overexpression plasmids, OSR2 overexpression plasmids, and the corresponding negative controls were transfected using lip2000 (Biosharp, City, Country, BL623B) according to the manufacturer’s instructions.

Western blotting

Total proteins were extracted with RIPA buffer containing protease inhibitors. Protein samples were separated by SDS-PAGE, transferred onto PVDF membranes, blocked, and incubated with primary antibodies against cleaved-caspase3, caspase3, cleaved-PARP, PARP, and GAPDH. HRP-conjugated secondary antibodies were applied, and signals were visualized using enhanced chemiluminescence (ECL).

Apoptosis assay

Cell apoptosis was quantified using an Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences, City, Country). After staining, cells were analyzed by flow cytometry (BD FACSCalibur), and apoptotic fractions were calculated using FlowJo software.

Luciferase reporter assay

Promoter fragments of WT1-AS containing predicted OSR2 binding motifs were cloned into pGL3-basic vectors. Cells were co-transfected with the reporter constructs and OSR2 overexpression plasmids. Firefly and Renilla luciferase activities were measured 48 h later using the Dual-Luciferase Reporter Assay System.

Xenograft mouse model

Female BALB/c nude mice (4–6 weeks old) were used to establish a subcutaneous xenograft model. HeLa cells stably overexpressing WT1-AS (WT1-AS-OE) and corresponding vector control cells were generated before implantation by plasmid transfection followed by puromycin selection. Prior to injection, cells were cultured under standard conditions and harvested during the logarithmic growth phase. After trypsinization, cells were washed twice with sterile phosphate-buffered saline (PBS), and cell viability was assessed by trypan blue exclusion. Only cell suspensions with viability >90% were used for implantation. A total of 1×10⁷ viable HeLa cells suspended in 0.1 mL of cell suspension were injected subcutaneously into the flank of each mouse. Tumor growth was monitored every 3 days, and tumor volume was calculated as (length × width²)/2. At the endpoint, tumors were harvested, weighed, and subjected to further histological analysis. Expression of apoptosis-related proteins, including cleaved-caspase-3, caspase-3, cleaved-PARP, and PARP, was examined by immunohistochemistry. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. All animal experiments were reviewed and approved by the Xinjiang Medical University Laboratory Animal Ethics Committee (No. IACUC-20240522-06), and were performed in accordance with institutional guidelines for the care and use of laboratory animals.

Statistical analysis

All statistical procedures were performed using R software (v4.2.1). Group comparisons were made using Student’s t-test or one-way analysis of variance (ANOVA), depending on the number of groups. Survival associations were examined with Kaplan-Meier and Cox regression models. Pearson correlation coefficients were calculated to assess relationships between continuous variables, with |r|≥0.3 considered biologically meaningful.

Results

Pan-cancer expression landscape and prognostic significance of WT1-AS

To characterize the transcriptional profile of WT1-AS across human cancers, we performed a systematic analysis using TCGA datasets covering 33 malignancies. Expression levels of WT1-AS varied considerably among tumor types. In several cancers, including lung adenocarcinoma (LUAD), colon adenocarcinoma (COAD), stomach adenocarcinoma (STAD) and prostate adenocarcinoma (PRAD), tumor samples exhibited significantly higher WT1-AS expression compared with matched normal tissues (Figure 2A, P<0.05). By contrast, some cancer types showed downregulation, including cervical squamous cell carcinoma (CESC), uterine corpus endometrial carcinoma (UCEC), and kidney renal papillary cell carcinoma (KIRP), indicating tumor-specific expression patterns.

Figure 2 Pan-cancer expression and prognostic significance of WT1-AS. (A) Boxplots showing differential expression of WT1-AS between tumor and corresponding normal tissues across 33 cancer types from TCGA. (B) Bubble plot summarizing the prognostic impact of WT1-AS expression in pan-cancer cohorts. Red and blue circles represent risk and protective factors, respectively, across DFI, DSS, PFI, and OS. The size of each circle corresponds to the −log₁₀ (P value). *, P<0.05; **, P<0.01; ***, P<0.001; ****, . DFI, disease-free interval; DSS, disease-specific survival; OS, overall survival; PFI, progression-free interval; TCGA, The Cancer Genome Atlas.

We next evaluated the prognostic significance of WT1-AS across TCGA cohorts. Survival analysis revealed that elevated WT1-AS expression was associated with poorer outcomes in multiple cancers, including kidney renal clear cell carcinoma (KIRC), brain lower grade glioma (LGG), and STAD (Figure 2B). Interestingly, in a subset of tumors such as ovarian cancer (OV) and mesothelioma (MESO), higher WT1-AS levels were correlated with improved prognosis, suggesting a context-dependent role. Collectively, these results highlight WT1-AS as a dysregulated lncRNA with diverse prognostic implications across different malignancies.

Association between WT1-AS expression and clinicopathological features in CESC

We next explored the relationship between WT1-AS expression and clinical parameters in CESC patients from TCGA-CESC. Stratified analyses based on age, pathologic T stage, lymph node (N) and distant metastasis (M) status, overall stage, and survival status revealed no statistically significant associations (Figure S1). Specifically, WT1-AS expression did not differ between younger (<60 years) and older (≥60 years) patients, nor among different T, N, or M categories. Similarly, tumor stage and vital status showed no significant correlation with WT1-AS levels. These results indicate that WT1-AS expression is not significantly associated with conventional clinicopathological parameters in the TCGA-CESC cohort, and therefore its clinical relevance in CESC should be interpreted with caution.

Functional characterization of WT1-AS in CESC

To further investigate the biological role of WT1-AS in CESC, we divided patients into high- and low-expression groups and identified DEGs. As shown in the volcano plot (Figure 3A), numerous genes were significantly dysregulated between the two groups, with a large proportion being upregulated in the WT1-AS-high cohort.

Figure 3 Differential expression and functional enrichment analysis of WT1-AS. (A) Volcano plot showing differentially expressed genes between WT1-AS high and low expression groups. The dashed line represents the threshold for statistical significance. (B) GO enrichment analysis of DEGs in WT1-AS high vs. low groups. The size of each dot represents the gene count, and color intensity corresponds to the −log₁₀ (P value). (C) KEGG pathway analysis of DEGs. The color scale indicates the −log₁₀ (P value) for each pathway. BP; CC, DEGs, differentially expressed genes; ECM, extracellular matrix; FC, fold change; GO, Gene Ontology; IL, interleukin; KEGG, Kyoto Encyclopedia of Genes and Genomes; MF.

GO enrichment analysis demonstrated that these DEGs were predominantly enriched in extracellular matrix (ECM) organization, structural constituents of the ECM, collagen fibril formation, humoral immune response, and ossification (Figure 3B). The cellular component terms were particularly enriched in collagen-containing ECM, basement membrane, and endoplasmic reticulum lumen, while the molecular function terms included glycosaminoglycan binding, integrin binding, and sulfur compound binding.

KEGG pathway analysis revealed that genes associated with WT1-AS were mainly involved in cancer-related signaling cascades, including PI3K-Akt and MAPK signaling, focal adhesion, Wnt signaling, ECM-receptor interaction, and cytokine-cytokine receptor interaction (Figure 3C). Additionally, enrichment was observed in immune- and infection-related pathways such as interleukin (IL)-17 signaling, rheumatoid arthritis, and Staphylococcus aureus infection.

Single-cell expression profile of WT1-AS in CESC

Single-cell transcriptomic data from the GSE168652 dataset were analyzed to further characterize the cellular distribution of WT1-AS in CESC. Cells were clustered into major lineages, including malignant epithelial cells, fibroblasts, endothelial cells, smooth muscle cell (SMC), CD8⁺ T cells, and monocytes/macrophages (Figure 4A). Visualization of WT1-AS expression demonstrated that its transcript levels were relatively low overall, with detectable expression mainly in fibroblasts and SMC rather than in malignant epithelial clusters (Figure 4B). Differential analysis between tumor and normal tissues confirmed that WT1-AS expression was significantly downregulated in malignant cells, while no significant changes were observed in immune or stromal subsets (Figure 4C).

Figure 4 Single-cell expression profile of WT1-AS in CESC. (A) UMAP plot showing the cellular distribution of different cell types in CESC (TCGA-CESC) based on single-cell RNA sequencing data (GSE168652). (B) UMAP plot depicting the expression levels of WT1-AS across the different cell types. (C) Violin plots showing the expression of WT1-AS in various cell types and comparing tumor vs. normal tissues. CESC, cervical cancer; SMC, smooth muscle cell; TCGA, The Cancer Genome Atlas; UMAP.

Identification of upstream transcription factors regulating WT1-AS

To explore the transcriptional regulation of WT1-AS, we first integrated FIMO motif predictions from the JASPAR database with correlation analysis in the TCGA-CESC cohort. As shown in the Venn diagram (Figure 5A), 10 overlapping genes were identified as candidate regulators of WT1-AS. Further intersection with prognosis-associated genes yielded three transcription factors: ZNF610, OSR2, and MEF2C (Figure 5B).

Figure 5 Identification and validation of OSR2 as an upstream regulator of WT1-AS. (A) Venn diagram showing the overlap between potential WT1-AS upstream regulators identified by FIMO motif analysis from the JASPAR database (green) and genes correlated with WT1-AS expression in TCGA-CESC (orange). (B) Venn diagram illustrating the intersection between WT1-AS target genes (red) and prognostic factors (blue) from the TCGA cohort. (C) Correlation analysis showing a positive correlation between WT1-AS and OSR2 expression in TCGA-CESC samples. (D) Kaplan-Meier survival analysis indicating that high expression of OSR2 is associated with better overall survival in CESC patients. CESC, cervical cancer; FIMO; JASPAR, OS, overall survival; TCGA, The Cancer Genome Atlas.

Correlation analysis demonstrated that WT1-AS expression was positively correlated with OSR2 levels (R=0.27, P=9.7e−07, Figure 5C). Motif enrichment and JASPAR-based binding site predictions further supported the potential binding of OSR2 to the WT1-AS promoter region (Table 1). Notably, survival analysis indicated that high OSR2 expression was significantly associated with improved OS in CESC patients (Figure 5D, P<0.001).

WT1-AS promotes apoptosis in CESC cells

To examine the functional role of WT1-AS in CESC, we established stable cell lines with WT1-AS overexpression (WT1-AS-OE) and corresponding vector controls (Figure 6A). Functional assays revealed that enforced expression of WT1-AS significantly increased the proportion of apoptotic cells, as determined by flow cytometry (Figure 6B,6C). Consistently, Western blotting of apoptosis-related proteins showed that WT1-AS overexpression led to increased levels of cleaved-caspase-3 and cleaved-PARP, whereas total caspase-3 remained unchanged and total PARP showed a slight reduction relative to controls (Figure 6D,6E).

Figure 6 Effect of WT1-AS overexpression on apoptosis and apoptotic protein expression in CESC cells. (A) Western blot analysis showing the overexpression of WT1-AS in CESC cells stably transfected with WT1-AS-OE or control vector (WT1-AS-NC). (B,C) Flow cytometric analysis of apoptosis in HeLa cells (B) and SiHa cells (C) transfected with WT1-AS overexpression plasmid (WT1-AS-OE) or negative control (WT1-AS-NC). (D,E) Western blot analysis of apoptosis-related proteins in HeLa cells (D) and SiHa cells (E) following WT1-AS overexpression. ns; *; ***, ****. CESC, cervical cancer; FITC; NC, OE, PARP; PI.

WT1-AS suppresses tumor growth and enhances apoptosis in vivo

To validate the effects of WT1-AS in vivo, a subcutaneous xenograft model was established using CESC cells stably transfected with either vector or WT1-AS-OE constructs. Tumor growth was monitored over time, and mice in the WT1-AS-OE group developed significantly smaller tumors compared with the vector group, indicating that WT1-AS overexpression impaired tumor growth (Figure 7A). Immunohistochemical staining of excised tumor tissues further confirmed elevated WT1-AS expression in the overexpression group. Consistent with the in vitro findings, the WT1-AS-OE tumors exhibited higher levels of cleaved-caspase-3 and cleaved-PARP, while total caspase-3 remained unchanged and PARP showed a modest decrease relative to controls (Figure 7B).

Figure 7 WT1-AS overexpression suppresses tumor growth and promotes apoptosis in vivo. (A) Representative images of xenograft tumors derived from WT1-AS-NC and WT1-AS-OE CESC cells in nude mice (left). Tumor weights were significantly reduced in the WT1-AS-OE group compared with the control (right). (B) Representative IHC staining and quantification of apoptosis-related proteins in xenograft tumor tissues. Positive staining regions are indicated by boxed areas to facilitate visualization of representative signals. ns, *, ****, P<0.0001. CESC, cervical cancer; IHC, immunohistochemistry; NC; OE; PARP.

OSR2 regulates WT1-AS and enhances its pro-apoptotic effects

Based on transcription factor prediction databases and correlation analysis in the TCGA-CESC cohort, OSR2 was identified as a potential upstream regulator of WT1-AS. Luciferase reporter assays confirmed the direct interaction between OSR2 and the WT1-AS promoter, supporting a regulatory relationship (Figure 8A).

Figure 8 OSR2 enhances the pro-apoptotic effects of WT1-AS in cervical cancer cells. (A) Dual-luciferase reporter assay showing the interaction between OSR2 and the WT1-AS promoter. (B) Flow cytometry analysis of apoptosis in cervical cancer cells with different transfections. (C) Western blot analysis of apoptosis-related proteins. (D) ns; ****. F/R, FITC, NC; OE; PARP; PI.

To further validate this mechanism, CESC cells were engineered to overexpress OSR2 (OSR2-OE) alone or in combination with WT1-AS (OSR2-OE + WT1-AS-OE). Flow cytometric analysis demonstrated that OSR2 overexpression increased the proportion of apoptotic cells compared with vector controls, and this effect was further amplified when OSR2 and WT1-AS were co-expressed (Figure 8B). Consistently, Western blotting revealed that OSR2-OE cells exhibited elevated levels of cleaved-caspase-3 and cleaved-PARP, while total caspase-3 remained unchanged and PARP showed a slight reduction relative to controls. Notably, co-overexpression of OSR2 and WT1-AS further enhanced cleaved-caspase-3 and cleaved-PARP expression compared with OSR2 alone, accompanied by increased total caspase-3 and reduced PARP levels (Figure 8C).

To determine whether WT1-AS mediates the pro-apoptotic effect of OSR2, WT1-AS knockdown was introduced into OSR2-overexpressing cells. In HeLa cells, silencing WT1-AS weakened the apoptosis-promoting effect of OSR2, as shown by a reduced apoptotic fraction and lower levels of cleaved-caspase-3 and cleaved-PARP compared with OSR2 overexpression alone (Figure 8B,8D). Similar findings were observed in SiHa cells, in which OSR2 overexpression increased apoptosis and apoptosis-related protein cleavage, whereas WT1-AS knockdown partially reversed these changes (Figure S2A,S2B). These results support that OSR2 promotes apoptosis, at least in part, through WT1-AS-dependent regulation, while also suggesting that OSR2 may exert additional WT1-AS-independent effects.

Discussion

In this study, we combined transcriptomic analyses with functional experiments to characterize the biological relevance of WT1-AS in CESC and to explore its upstream transcriptional regulation by OSR2. Our findings revealed that WT1-AS is significantly dysregulated across multiple tumor types and shows associations with survival outcomes in pan-cancer analyses. Importantly, in CESC, WT1-AS functions as a tumor suppressor by promoting apoptosis, and its transcriptional activation is mediated by OSR2. These results highlight the WT1-AS/OSR2 regulatory axis as a novel mechanism in CESC progression.

However, in the TCGA-CESC cohort, WT1-AS expression was not significantly associated with conventional clinicopathological parameters. Therefore, although WT1-AS may have biological and possible prognostic relevance at the transcriptomic level, the current evidence is insufficient to support its direct clinical applicability as a standalone prognostic biomarker in CESC.

Our pan-cancer analysis demonstrated that WT1-AS expression is altered in a wide range of malignancies, with divergent prognostic implications. This is consistent with the emerging view that lncRNAs often exert context-dependent functions, acting as oncogenes in some cancer types while serving as tumor suppressors in others (35,36). For example, WT1-AS has been reported to inhibit proliferation and invasion in gastric cancer (37) and breast cancer (27), whereas in acute myeloid leukemia, it has been implicated in leukemogenesis through interaction with WT1 (38,39). Our study extends these observations to CESC, where WT1-AS exerts pro-apoptotic and tumor-suppressive effects.

One point that merits careful interpretation is the apparent difference between the single-cell transcriptomic analysis and the in vitro functional data. In the single-cell dataset, WT1-AS expression was relatively low in malignant epithelial cells, whereas enforced WT1-AS expression in HeLa and SiHa cells promoted apoptosis. These findings are not necessarily contradictory. Low endogenous expression in malignant cells may itself be consistent with a tumor-suppressive role, as downregulation of WT1-AS could represent one mechanism by which CESC cells evade apoptosis. In this context, overexpression experiments do not imply that WT1-AS is naturally abundant in malignant cells; rather, they demonstrate the functional consequence of restoring WT1-AS expression. In addition, differences between patient-derived single-cell data and established cell lines may reflect cellular adaptation, clonal selection, and the absence of stromal or microenvironmental influences in vitro. Therefore, the low WT1-AS expression observed in malignant epithelial cells and the pro-apoptotic effect induced by WT1-AS restoration in cultured cells may represent complementary, rather than conflicting, aspects of its tumor-suppressive role.

In CESC, several lncRNAs such as HOTAIR and MALAT1 have been widely studied and are known to promote tumor progression through pathways including epithelial-mesenchymal transition and apoptosis suppression (40-46). Compared with these oncogenic lncRNAs, our findings indicate that WT1-AS may function oppositely as a protective factor, promoting apoptosis via the activation of caspase signaling. Indeed, our functional assays confirmed that overexpression of WT1-AS significantly increased levels of cleaved-caspase3 and cleaved-PARP, both of which are critical markers of apoptosis. These results are in line with prior studies showing that lncRNAs can regulate caspase activation and mitochondrial apoptosis pathways (47-50), but to our knowledge, this is the first report directly linking WT1-AS to apoptotic regulation in CESC.

Furthermore, we identified OSR2 as an upstream transcriptional regulator of WT1-AS. OSR2, a transcription factor primarily studied in developmental biology and osteogenic differentiation (33,51-53), has not been extensively investigated in cancer. Recent bioinformatics studies have hinted at its potential tumor-suppressive role (34,54). Our results suggest that OSR2 enhances apoptosis, at least in part, by transcriptionally activating WT1-AS. The observation that co-overexpression of OSR2 and WT1-AS further strengthened apoptotic changes supports a functional relationship between the two molecules, whereas the partial attenuation of OSR2-induced apoptosis after WT1-AS knockdown indicates that WT1-AS is an important, but likely not exclusive, downstream mediator of OSR2. This interpretation is biologically plausible, as transcription factors such as OSR2 may regulate multiple downstream targets in parallel.

Although the increased levels of cleaved-caspase-3 and cleaved-PARP support a pro-apoptotic role of WT1-AS in CESC, the downstream mechanism underlying this effect remains insufficiently defined. Previous studies in CESC have suggested that WT1-AS may regulate tumor progression through p53-associated signaling, including a reported miR-330-5p/p53 regulatory axis (30), raising the possibility that the pro-apoptotic effect of WT1-AS may be mediated, at least in part, through miRNA-dependent regulation. In addition, as a lncRNA, WT1-AS may also influence apoptosis through interactions with RNA-binding proteins, transcriptional regulators, or chromatin-associated complexes. Our enrichment analyses further suggest that WT1-AS may participate in broader survival-related pathways, including PI3K-Akt and MAPK signaling. These mechanistic possibilities remain to be validated experimentally, and further studies will be needed to clarify the precise downstream effectors through which WT1-AS promotes apoptosis in CESC.

Despite these significant findings, several limitations must be acknowledged. First, the bioinformatic analyses were based mainly on retrospective public datasets, which may be affected by sample heterogeneity, incomplete clinical annotation, and selection bias. Second, although our functional experiments support a pro-apoptotic role for WT1-AS and identify OSR2 as an upstream regulator, the downstream molecular mechanism linking WT1-AS to apoptotic signaling remains insufficiently resolved. Third, while our conclusions were strengthened by validation in both HeLa and SiHa cells, these models still represent only a limited part of the biological heterogeneity of CESC and cannot fully recapitulate the complexity of primary tumors or the tumor microenvironment. Finally, the translational relevance of the WT1-AS/OSR2 axis will require further confirmation in larger clinical cohorts and in more physiologically relevant models, such as patient-derived organoids or in vivo systems with greater tumor heterogeneity.

Conclusions

In conclusion, our study supports a tumor-suppressive role of WT1-AS in CESC and identifies OSR2 as a previously unrecognized upstream transcriptional regulator. Together, these findings expand current understanding of WT1-AS biology in CESC, while also indicating that its mechanistic basis and translational relevance require further investigation.

Acknowledgments

The authors acknowledge the use of Nano Banana software (an AI-powered illustration model) for generating Figure 1 based on the experimental data obtained in this study. All elements in the figure are original and were created solely by the authors.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2842/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2842/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2842/prf

Funding: This work was supported by the Department of Science and Technology of Xinjiang Uygur Autonomous Region, General Program of Natural Science Foundation (No. 2024D01C189).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2842/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. All animal experiments were reviewed and approved by the Xinjiang Medical University Laboratory Animal Ethics Committee (No. IACUC-20240522-06), and were performed in accordance with institutional guidelines for the care and use of laboratory animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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