TCEAL5 cooperates with the NuRD complex to epigenetically silence mesenchymal genes in glioma

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This study analyzed TCEAL5 expression in glioma and investigated its biological functions through cell assays and molecular analyses. Our findings revealed a significant reduction in TCEAL5 expression in glioma tissues, with lower expression levels correlating with higher histologic grades and poorer prognosis. Further experimental investigations demonstrated that ectopic overexpression of TCEAL5 in glioma cell lines significantly inhibited cell migration and invasion. Mechanistic studies indicated that TCEAL5 exerts its inhibitory effects on EMT by directly binding to the promoters of mesenchymal genes. Additionally, TCEAL5 was found to interact with the NuRD complex, leading to transcriptional repression of mesenchymal genes via epigenetic modulation. These findings highlight the multifaceted role of TCEAL5 as a tumor suppressor in glioma, suggesting its potential as a prognostic biomarker and a target for therapeutic intervention. Our study not only adds to the understanding of TCEAL5's biological functions but also opens new avenues for research into its application in cancer therapy. TCEAL5 NuRD EMT Glioma Tumor suppressor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction The TCEAL5 gene is a member of the TCEAL family of genes, which encode proteins containing TFA domains that might modulate transcription in a promoter context-dependent manner. TCEAL1, a TCEAL family member, is reported to be a nuclear phosphoprotein functioning to repress Rous sarcoma virus long-terminal repeat transcription and to contribute to preventing the cellular transformation induced by the Rous sarcoma virus long-terminal repeat 1 . On the other hand, the closely related protein transcription elongation factor SII (TFIIS/TCEA1) has been reported to be implicated in transcription elongation and transcript fidelity 2 , 3 . The TCEAL gene family is implicated in various biological processes and diseases. TCEAL1 is associated with X-linked dominant neurodevelopmental syndromes and influences neurological traits 4 . TCEAL2 has been highlighted as a prognostic marker in pan-cancer studies, indicating its significance in cancer biology 5 . TCEAL2 has also been reported to function as a tumor suppressor in renal cell carcinoma and to correlate with better patient prognosis 6 . Furthermore, TCEAL7 is recognized as a potential tumor suppressor gene, with a particular role in negatively regulating the NF-kappaB pathway, and is involved in the regulation of c-Myc activity in alternative lengthening of telomeres 7 – 9 . The diverse functions of the TCEAL gene family members underline their importance in both development and disease. Compared to other TCEAL family members, TCEAL5 remains a relatively understudied member of the TCEAL gene family. While scarce, emerging evidence suggests TCEAL5's roles in cellular proliferation, cardiac health, and muscle cell differentiation 10 – 13 . In the present study, we report that TCEAL5 expression is decreased in glioma and forced expression of TCEAL5 inhibits cell migration and invasion. Mechanistic study demonstrates that TCEAL5 interacts with the NuRD complex to repress mesenchymal marker genes expression, thus to inhibit epithelial-mesenchymal transition (EMT) in glioma. These results indicate that TCEAL5 may function as a tumor repressor in human glioma. 2. Materials and methods 2.1 Data source and statistical analysis Data on mRNA expressions and the clinical traits of human glioma samples and normal samples were obtained from the TCGA (The Cancer Genome Atlas Program (TCGA)), CGGA (Chinese Glioma Genome Atlas) and GTEx (Genotype-Tissue Expression) initiatives. The mRNA expression datasets underwent processing via the UCSC Toil RNAseq Recompute approach, ensuring a unified analysis across various datasets devoid of batch effects ( https://xenabrowser.net/hub/ ). Statistical evaluations were performed utilizing the R software, version 4.2.0 ( http://www.r-project.org/ ). To explore TCEAL5's clinical relevance in glioma, its expression levels were categorized into high and low groups, based on an optimal cutoff value determined by the surv_cutoff function of the survminer R package. The log-rank test was employed to compare survival curves across these groups. 2.2 Cell lines and cell culture U251 cells and Ln229 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), which performs routine cell line authentication testing with short tandem repeat analysis. Mycoplasma contamination was regularly examined and no contamination occurred during this study. Cells were cultured as previously described 14 . TCEAL5 cDNA was cloned into the GV492 lentivirus vector (GeneChem, China) to enable ectopic expression in human glioma cells. Lentiviruses were produced by the transfection of 293T cells with plasmids using the packaging Mix (GeneChem, China). For TCEAL5 ectopic expression, glioma cells were infected with the lentiviruses and selected with puromycin. 2.3 RNA extraction and reverse transcription-quantitative PCR (RT-qPCR) Cells were subjected to total RNA extraction using Invitrogen's Trizol reagent (Cat. No. 15596026), adhering strictly to the provided guidelines. Post-extraction, the integrity and concentration of the RNA were assessed through gel electrophoresis and measured using the Thermoscientific Nanodrop2000 spectrophotometer. To eliminate genomic DNA contamination and synthesize cDNA, 2 µg of the RNA underwent reverse transcription utilizing Vazyme's HiScript III RT SuperMix for qPCR (+ g DNA wiper) (Cat. No. R123-01), in compliance with the supplier's protocol. The synthesized cDNA was then analyzed using quantitative real-time PCR (qPCR) on the ABI-7500 system, employing TAKARA's TB Green™ Premix Ex Taq™ (Tli RNaseH Plus) (Cat. No. RR420A). β-actin was employed as the internal control for normalization. 2.3 Western blot and antibodies The cells were processed for protein extraction by lysing them in RIPA (150 mM NaCl, 2 mM EDTA, 50 mM Tris pH 8.0, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail). Post-lysis, the lysate was centrifuged at 12,000 g and 4°C for 20 minutes. The protein content of the supernatant was then quantified using the Bradford protein assay. The whole cell extracts were subjected to SDS-PAGE, and were subsequently transferred onto a PVDF membrane. The membrane was then blocked using a 5% solution of nonfat dry milk in TBST to prevent non-specific binding. This was followed by incubation with primary antibodies overnight at 4°C, and then with HRP-conjugated secondary antibodies at room temperature for 2 hours. Finally, the chemiluminescence reaction was carried out according to the manufacturer's instructions. The primary antibodies used in Western blot are as follows: anti-FLAG (Sigma, F3165), anti-E-cadherin (Proteintech, 20874-1-AP), anti-N-cadherin (Proteintech, 22018-1-AP), anti-TWIST1 (Proteintech, 25465-1-AP), anti-MTA2 (Proteintech, 17554-1-AP), anti-HDAC1 (Proteintech, 10197-1-AP), anti-CHD4 (Proteintech, 14173-1-AP), anti-β-tubulin (Proteintech, 66240-1-Ig). 2.4 Immunofluorescence and antibodies Cells, cultivated on glass coverslips placed in 24-well plates, underwent fixation using a 4% solution of formaldehyde and were then made permeable with a PBS solution infused with 0.2% Triton X-100. A blocking step was conducted using PBS mixed with 5% bovine serum albumin for one hour, followed by a room temperature incubation with the primary antibody for another hour. This was succeeded by a one-hour incubation with secondary antibodies conjugated to a fluorescent dye, and subsequent DAPI staining. Microscopic imaging of the cells was carried out using a Leica microscope setup. The following antibodies were used in immunofluorescence: anti-FLAG (Sigma, F3165), anti-E-cadherin (Proteintech, 20874-1-AP), anti-N-cadherin (Proteintech, 22018-1-AP), anti-TWIST1 (Proteintech, 25465-1-AP). 2.5 Wound healing assays and transwell cell invasion assays In the wound-healing assay, cells were cultured in 6-well plates. An overnight serum starvation was initiated, post which a sterile 1ml pipette tip was used to create a scratch, simulating a wound in the cell monolayer. After removing the dislodged cells with a PBS rinse, the culture was continued in a medium enriched with 1% FBS. The healing process, particularly the closure of the gap, was monitored and documented at specified time points using a light microscope. For the transwell in vitro cell invasion assays, cells, approximately 2×10 5 , were placed in the Matrigel pre-coated top chamber of the transwell chambers (8.0 mm, Corning), while the lower wells received complete medium to act as a chemoattractant and encourage cell invasion. The setup was then incubated for a period ranging from 24 to 48 hours within a cell culture incubator. Following incubation, cells that remained on the upper side of the membrane were carefully wiped away with a cotton swab. The cells that successfully migrated and adhered to the bottom side of the membrane were then stained with 0.1% Crystal Violet. The quantification of these cells was carried out manually to assess the invasion capability. 2.6 Cell proliferation assay and clonogenic assay Cell proliferation was assessed employing the Cell Counting Kit-8 (Beyotime, C0038), strictly in line with the instructions provided by the manufacturer. The procedure involved seeding cells at a density of 3×10 3 cells per well in 96-well plates. After the seeding, 100µl of serum-free culture medium, supplemented with 10µl of WST-8 reagent, was introduced to the cells at pre-determined time intervals. The plates were then placed in a cell culture incubator and allowed to incubate for 2 hours. Post-incubation, the optical absorbance of each well was measured at a wavelength of 450nm using a microplate reader to evaluate cell proliferation. In parallel, clonogenic assays were executed to examine the colony-forming ability of the cells. This involved seeding cells in 6-well plates at a density of 1000 cells per well. The seeded cells were incubated in a cell culture incubator for a duration of 14 days, allowing colonies to form. At the end of the incubation period, cells were stained using crystal violet staining solution (Beyotime, C0121) and the colonies were counted manually to quantify the clonogenic potential of the cells. 2.7 Immunoprecipitation To conduct co-immunoprecipitation experiments, cells were lyzed using lysis buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, protease inhibitor cocktail) for 20 minutes at 4°C. Subsequent centrifugation at 14,000 g for 15 minutes at 4°C followed. The resulting protein supernatant was then incubated with 2 µg of specific antibodies for 12 hours at 4°C under constant rotation. Afterwards, 50 µl of a 50% solution of protein A or G agarose beads was added, extending the incubation for an additional 2 hours. The beads were washed five times with the original lysis buffer, with centrifugation at 500 g for 3 minutes at 4°C to collect the beads between washes. Proteins bound to the beads were eluted by resuspending in 2x SDS PAGE loading buffer and heated for 5 minutes. To identify the TCEAL5-interacting proteins, the eluates were then separated on a SDS-PAGE gel, silver stained, and analyzed through mass spectrometry. 2.8 Chromatin immunoprecipitation Cells underwent crosslinking with 1% formaldehyde for 10 minutes at room temperature, which was then quenched by adding glycine to achieve a final concentration of 125 mM for 5 minutes. Subsequently, the cells were prepared in lysis buffer (1% SDS, 5 mM EDTA, and 50 mM Tris-HCl pH 8.0, protease inhibitors). This mixture was then exposed to cycles of sonication for 30 seconds on and off using a Bioruptor, resulting in chromatin fragments approximately 300 bp long. The lysates were then diluted in Dilution Buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl pH 8.0, protease inhibitors). For immunoprecipitation (IP), this diluted chromatin was incubated either with normal IgG as a control or with specific antibodies for 12 hours at 4°C under continuous rotation. Following this, 50 µL of 50% (volume/volume) protein A/G Sepharose beads were added, and the incubation continued for an additional 2 hours. The beads were then washed using wash buffers sequentially: Wash buffer I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, and 20 mM Tris-HCl, pH 8.0); Wash buffer II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, and 20 mM Tris-HCl, pH 8.0); Wash buffer III (0.25 M LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA, and 10 mM Tris-HCl, pH 8.0); and TE (1 mM EDTA and 10 mM Tris-HCl, pH 8.0). Finally, the chromatin complexes and input were de-crosslinked at 55°C for 12 hours in elution buffer (1% SDS and 0.1 M NaHCO3), and the DNA was purified using the QIAquick PCR Purification Kit. The DNA was then analyzed using quantitative real-time PCR (qPCR) on the ABI-7500 system, employing TAKARA's TB Green™ Premix Ex Taq™ (Tli RNaseH Plus) (Cat. No. RR420A). 3. Results 3.1 TCEAL5 expression is significantly decreased in human glioma and higher expression favors prognosis To explore TCEAL5's role in human glioma, an initial examination of its expression levels was conducted by analyzing RNA-sequencing data from both the TCGA (The Cancer Genome Atlas) and GTEx (Genotype-Tissue Expression) datasets. The analysis revealed a significant decrease in TCEAL5 expression in glioma samples compared to normal tissue samples (Fig. 1 A). To further deepen the investigation, a survey of TCEAL5 expression levels across histologic grades was carried out. The results indicated a negative correlation between TCEAL5 expression and histologic grades, suggesting a potential inverse relationship between TCEAL5 expression and malignancy (Fig. 1 B). These findings collectively point towards the possible significance of TCEAL5 in the progression of glioma. To further explore TCEAL5's clinical relevance in glioma, its expression levels were categorized into high and low groups, based on an optimal cutoff value determined by the surv_cutoff function of the survminer R package. The log-rank test was employed to compare survival curves across these groups. As depicted in Fig. 1 C, a notable association was observed between higher TCEAL5 expression and improved overall survival rates in glioma patients, underscoring a potential prognostic significance of TCEAL5 expression. This association was further supported by analysis using datasets from the CGGA, reinforcing the observation that higher TCEAL5 expression might be indicative of a more favorable prognosis in glioma (Fig. D). Collectively, these findings suggest a pivotal role for TCEAL5 in the pathology of glioma and its potential as a biomarker for patient prognosis. 3.2 TCEAL5 inhibits glioma cell migration and invasion To investigate TCEAL5's regulatory influence on glioma, TCEAL5 was ectopically expressed in two glioma cell lines (Fig. 2 A). This intervention's impact on cell dynamics was assessed through Cell Counting Kit 8 (CCK8) analysis for cell proliferation and a colony formation assay to gauge the cells' capacity for clone formation, a potential indicator of tumor growth potential. As depicted in Supplementary Figure S1, the overexpression of TCEAL5 only led to minor alterations in cell proliferation and colony-forming ability in both cell lines. This outcome suggests TCEAL5's role in glioma cell biology likely involves other mechanisms. To further explore TCEAL5's role in glioma, the focus shifted to its influence on cell migration and invasion, critical factors in cancer metastasis. The wound healing assay revealed that the overexpression of TCEAL5 notably impeded the movement of glioma cells, as evidenced in Figs. 2 B and 2 C. This inhibitory effect on migration was further substantiated through a transwell cell invasion assay, which similarly demonstrated a marked reduction in the invasion capabilities of glioma cells upon TCEAL5 overexpression (Fig. 2 D and 2 E). Collectively, these findings underscore TCEAL5's role as a deterrent in the processes of glioma cell migration and invasion, highlighting its potential significance in mitigating the metastatic progression of the cancer. 3.3 TCEAL5 regulates EMT genes in glioma Based on the observation that TCEAL5 hinders glioma cell migration and invasion, it was hypothesized that TCEAL5 might execute this function by repressing epithelial-mesenchymal transition (EMT) in glioma. EMT is a critical process through which cancer cells acquire the ability to migrate and invade, often culminating in metastasis. To examine the impact of TCEAL5 overexpression on EMT gene expression, both western blot assays and immunofluorescence staining assays were employed. The findings indicated a decrease in mesenchymal gene expression alongside an increase in epithelial gene expression upon TCEAL5 overexpression, suggesting that TCEAL5 overexpression induces a mesenchymal-to-epithelial transition (MET)-like process in glioma cells (Fig. 3 A and 3 B; Supplementary Fig. S2). To validate the direct regulatory influence of TCEAL5 on EMT gene expression, chromatin immunoprecipitation assays were conducted. These assays confirmed the binding of TCEAL5 to the promoters of mesenchymal genes N-cadherin and TWIST1, while no TCEAL5 binding was observed at the promoter of the epithelial gene E-cadherin, as depicted in Fig. 3 C. This binding illustrates a direct regulatory mechanism of TCEAL5 on N-cadherin and TWIST1. In contrast, TCEAL5's regulation of E-cadherin likely involves indirect mechanisms. These findings collectively underscore the critical role of TCEAL5 in the regulation of EMT and potentially in the metastatic progression of glioma. 3.4 TCEAL5 interacts with the NuRD complex in glioma cells In an effort to decode the specific mechanisms through which TCEAL5 influences the expression of mesenchymal genes N-cadherin and TWIST1, co-immunoprecipitation assay was performed. This assay aimed to purify TCEAL5 and its interacting partners in glioma cells. Post-purification, the immunoprecipitation (IP) fractions underwent separation via SDS-PAGE, and the distinct bands observed in the gels were further analyzed using Mass spectrometry. The result showed that TCEAL5 was co-purified with multiple components of the NuRD complex, a chromatin remodeling complex, as depicted in Figs. 4 A and 4 B. The validity of this interaction was reinforced through reciprocal co-immunoprecipitation assays, the results of which are displayed in Fig. 4 C. This collection of evidence robustly suggests that TCEAL5 collaborates with the NuRD complex in glioma cells, pointing towards an intriguing regulatory relationship in the modulation of gene expression. 3.5 TCEAL5-mediated recruitment of the NuRD complex silences mesenchymal gene transcription Next, the investigation delved into whether TCEAL5's suppression of mesenchymal gene expression, specifically N-cadherin and TWIST1, is facilitated by the NuRD complex. Chromatin immunoprecipitation assays were utilized to assess the impact of TCEAL5 overexpression on the NuRD complex's presence at the promoters of these genes. The findings indicated an increase in the NuRD complex's occupancy at the N-cadherin and TWIST1 promoters, suggesting a recruitment mechanism by TCEAL5 (Fig. 5 A- 5 C). Given the NuRD complex's known association with transcriptional repression, largely due to the presence of HDAC1, the study further examined the potential consequences of this increased occupancy on histone acetylation at these promoters. The results revealed a reduction in the levels of H3K9ac and H3K27ac, histone marks typically associated with active transcription, upon TCEAL5 overexpression and corresponding increase in NuRD occupancy (Fig. 5 D and 5 E). This suggests a direct epigenetic modulation by TCEAL5, leading to the transcriptional repression of these mesenchymal genes. To solidify the understanding of TCEAL5's role at the transcriptional level, qRT-PCR was conducted to measure the mRNA levels of N-cadherin and TWIST1 following TCEAL5 overexpression. The results from this analysis aligned with the previous findings at the protein level, confirming that TCEAL5 indeed exerts an inhibitory influence on the transcription of N-cadherin and TWIST1 (Fig. 5 F). These findings collectively underscore the intricate regulatory role of TCEAL5 in glioma, particularly in its ability to modulate the expression of crucial genes involved in cell migration and invasion through epigenetic mechanisms. 4. Discussion TCEAL5 is a relatively understudied TCEAL gene family member. This study revealed that TCEAL5 expression is substantially decreased in human glioma, and this reduction is inversely correlated with the tumor malignancy, and higher TCEAL5 expression is associated with improved prognosis in glioma patients, underscoring its potential as a prognostic biomarker. Interestingly, despite the observed decline in TCEAL5 expression in glioma, its forced overexpression did not markedly alter cell proliferation or colony formation capabilities. This suggests that the influence of TCEAL5 extends beyond mere modulation of cell growth, a hypothesis substantiated by the subsequent observations of its role in impeding cell migration and invasion. These processes are critical in cancer metastasis, and the study's findings that TCEAL5 overexpression leads to reduced migration and invasion capabilities in glioma cells point to its potential role in mitigating metastatic progression.The mechanisms underlying TCEAL5's regulatory effects appear to involve the modulation of EMT gene expression. Our results demonstrated that TCEAL5 overexpression induces a MET-like process. The direct binding of TCEAL5 to the promoters of mesenchymal genes like N-cadherin and TWIST1, coupled with the indirect influence on the epithelial gene E-cadherin, reveals a nuanced regulatory mechanism through which TCEAL5 exerts its function. An intriguing aspect of TCEAL5's mechanism of action is its interaction with the NuRD complex, a chromatin remodeling complex known to influence gene expression. The study's findings suggest that TCEAL5 recruits the NuRD complex to the promoters of mesenchymal genes, leading to a reduction in histone acetylation and subsequent transcriptional repression of these genes. This epigenetic modulation offers a layer of complexity to the understanding of TCEAL5's role in glioma and underscores the potential for targeting these interactions in therapeutic interventions. In conclusion, this study sheds light on the pivotal role of TCEAL5 in glioma, presenting a comprehensive overview of its expression dynamics, regulatory mechanisms, and clinical implications. The insights gained pave the way for future research and potential therapeutic strategies, emphasizing the importance of TCEAL5 in the pathology of glioma and possibly other cancers. Abbreviations TCEAL5 transcription elongation factor A like 5 NuRD Nucleosome Remodeling Deacetylase complex EMT Epithelial-Mesenchymal Transition TCGA The Cancer Genome Atlas CGGA Chinese Glioma Genome Atlas. Declarations Acknowledgements We thank all the members in Dr. Zhang’s lab for insightful scientific discussion. Disclosure Funding Information This work was supported by the National Natural Science Foundation of China (81972653). Conflict of Interest The authors declare that they have no competing interests. Ethics Statement ―Approval of the research protocol by an Institutional Reviewer Board: N/A. ―Informed Consent: N/A. ―Registry and the Registration No. of the study/trial: N/A. ―Animal Studies: N/A. ―Author Contribution: Conceptualization: DZ; methodology: DZ, HZ, XL, YZ, QZ, XZ; validation: DZ, HZ, XL, YZ, QZ, XZ; investigation: DZ, HZ, XL, YZ, QZ, XZ; data curation: DZ, HZ; writing: DZ; visualization: DZ, HZ; supervision: DZ; funding acquisition: DZ. Data availability statement Data is provided within the manuscript or supplementary materials files. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3907845","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":290182189,"identity":"90b7a7ed-c568-4f46-854d-7f229d00a7c5","order_by":0,"name":"Hanchi Zhou","email":"","orcid":"","institution":"Xuzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hanchi","middleName":"","lastName":"Zhou","suffix":""},{"id":290182190,"identity":"ffee6dc2-aa9e-4fbd-b6dd-d936dcbc810a","order_by":1,"name":"Xue Li","email":"","orcid":"","institution":"Xuzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xue","middleName":"","lastName":"Li","suffix":""},{"id":290182191,"identity":"ff3ca1db-6312-4e2e-9255-0d719920fe98","order_by":2,"name":"Yirao Zhang","email":"","orcid":"","institution":"Xuzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yirao","middleName":"","lastName":"Zhang","suffix":""},{"id":290182192,"identity":"4a166888-b142-4382-b6a5-a4949819ef6e","order_by":3,"name":"Qian Zhang","email":"","orcid":"","institution":"Xuzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Zhang","suffix":""},{"id":290182193,"identity":"3ab9f91f-78e2-4686-95e5-64ef4d486435","order_by":4,"name":"Xinwei Zhou","email":"","orcid":"","institution":"Xuzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xinwei","middleName":"","lastName":"Zhou","suffix":""},{"id":290182194,"identity":"f02152c4-8665-406b-8f20-2fcd3f0f02e1","order_by":5,"name":"Daoyong Zhang","email":"data:image/png;base64,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","orcid":"","institution":"Xuzhou Medical University","correspondingAuthor":true,"prefix":"","firstName":"Daoyong","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-01-29 04:08:07","currentVersionCode":3,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-3907845/v3","doiUrl":"https://doi.org/10.21203/rs.3.rs-3907845/v3","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54598549,"identity":"e9f81da2-e520-4a5f-9e43-9a7b3fb5240a","added_by":"auto","created_at":"2024-04-12 20:10:58","extension":"tif","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":90343,"visible":true,"origin":"","legend":"\u003cp\u003eTCEAL5 expression was lower in glioma tissues and linked to disease advancement and patient survival. \u003cstrong\u003eA, B. \u003c/strong\u003eTCEAL5 expression analysis, utilizing TCGA and CGGA data, revealed a notable decrease in glioma compared to normal tissues (A), inversely correlating with histologic grade of malignancy (B). \u003cstrong\u003eC, D. \u003c/strong\u003eHigher TCEAL5 expression correlated with a better prognosis in glioma patients. The number of cases in each group is specified, with a p-value of \u0026lt;0.001 in the Log-rank test.\u003c/p\u003e","description":"","filename":"Figure12.tif","url":"https://assets-eu.researchsquare.com/files/rs-3907845/v3/d56368f6dc1aee9b1c61aa87.tif"},{"id":54598553,"identity":"6de46f86-9c79-4b92-b659-4630020a0572","added_by":"auto","created_at":"2024-04-12 20:10:59","extension":"tif","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1988831,"visible":true,"origin":"","legend":"\u003cp\u003eTCEAL5 inhibits glioma cell migration and invasion. \u003cstrong\u003eA. \u003c/strong\u003eEctopicTCEAL5 expression in glioma cells was confirmed through Western Blotting assays, using β-tubulin as the loading control. \u003cstrong\u003eB, C. \u003c/strong\u003eOverexpression of TCEAL5 impedes the migration of glioma cells. This was determined by conducting wound healing assays to evaluate the impact of heightened TCEAL5 levels on cell movement. \u003cstrong\u003eD, E. \u003c/strong\u003eHeightened TCEAL5 expression impedes the invasion of glioma cells. The impact on cell invasion due to increased TCEAL5 was assessed using Transwell invasion assays. Data from three separate experiments were quantified and presented as mean + S.D., with significant levels marked as **p\u0026lt;0.01 and ***p\u0026lt;0.001 in the unpaired t-test. The scale bar represents 100 μm.\u003c/p\u003e","description":"","filename":"Figure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-3907845/v3/5bab836e0b6b9a061468a807.tif"},{"id":54598548,"identity":"939ab18d-b062-40dc-b10f-cbc64f860f44","added_by":"auto","created_at":"2024-04-12 20:10:58","extension":"tif","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1123735,"visible":true,"origin":"","legend":"\u003cp\u003eTCEAL5 modulates the expression of EMT genes. \u003cstrong\u003eA, B. \u003c/strong\u003eOverexpression of TCEAL5 impacts EMT marker genes expression. Expression levels of EMT marker genes were measured by western blotting and immunofluorescence staining. Scale bar, 50 μm. \u003cstrong\u003eC.\u003c/strong\u003e TCEAL5 binds to the promoters of mesenchymal genes N-cadherin and TWIST1. Chromatin immunoprecipitation was utilized to ascertain TCEAL5's binding at the promoter regions of N-cadherin and TWIST1 mesenchymal genes. Data from three individual experiments were consolidated, quantified, and depicted as mean + S.D., with significance indicated by ***p\u0026lt;0.001 in the unpaired t-test.\u003c/p\u003e","description":"","filename":"Figure32.tif","url":"https://assets-eu.researchsquare.com/files/rs-3907845/v3/08ee948edf22ca4e9edc4695.tif"},{"id":54598552,"identity":"c40f53b5-0249-4ffb-9c0f-f10a61579aa2","added_by":"auto","created_at":"2024-04-12 20:10:58","extension":"tiff","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1177127,"visible":true,"origin":"","legend":"\u003cp\u003eTCEAL5 interacts with the NuRD complex in glioma cells. \u003cstrong\u003eA. \u003c/strong\u003eCell extracts from cells expressing TCEAL5-FLAG were subjected to immunoprecipitation (IP) using anti-FLAG antibodies, with extracts from cells expressing FLAG alone serving as controls. The IP fractions were then separated using SDS-PAGE and visualized through silver staining. \u003cstrong\u003eB, C.\u003c/strong\u003e Reciprocal co-immunoprecipitation assays were conducted to confirm the interaction between TCEAL5 and the NuRD complex within glioma cells.ocal co-immunoprecipitation assays were conducted to confirm the interaction between TCEAL5 and the NuRD complex within glioma cells.\u003c/p\u003e","description":"","filename":"Figure42.tiff","url":"https://assets-eu.researchsquare.com/files/rs-3907845/v3/c9acd04c43c1d297411c00dc.tiff"},{"id":54598550,"identity":"729a805b-5f2f-4708-b5ab-56ebbb67f108","added_by":"auto","created_at":"2024-04-12 20:10:58","extension":"tiff","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":30456,"visible":true,"origin":"","legend":"\u003cp\u003eTCEAL5-mediated recruitment of the NuRD complex silences mesenchymal gene transcription. \u003cstrong\u003eA-C. \u003c/strong\u003eOverexpression of TCEAL5 enhances the NuRD complex's presence at the promoters of mesenchymal genes N-cadherin and TWIST1. Chromatin immunoprecipitation was employed to evaluate how TCEAL5 overexpression influences the NuRD complex's binding to the promoter of these mesenchymal genes. \u003cstrong\u003eD, E. \u003c/strong\u003eOverexpression of TCEAL5 decreases histone acetylation at the promoters of mesenchymal genes N-cadherin and TWIST1. \u003cstrong\u003eF. \u003c/strong\u003eOverexpression of TCEAL5 suppresses the transcription of mesenchymal genes N-cadherin and TWIST1.\u003c/p\u003e","description":"","filename":"Figure5.tiff","url":"https://assets-eu.researchsquare.com/files/rs-3907845/v3/2288127d5e2cb95634ce07ef.tiff"},{"id":54598554,"identity":"2c29bb3a-63e0-4343-9592-8182f7ac6a6b","added_by":"auto","created_at":"2024-04-12 20:11:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5437007,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3907845/v3/3b37d5a9-b8f5-4ca5-ab8a-0b23f57dd565.pdf"},{"id":54598551,"identity":"8016da42-7b1c-4857-8e66-1488b4b3694e","added_by":"auto","created_at":"2024-04-12 20:10:58","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":413485,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3907845/v3/01619284269576ead9572215.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"TCEAL5 cooperates with the NuRD complex to epigenetically silence mesenchymal genes in glioma","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe TCEAL5 gene is a member of the TCEAL family of genes, which encode proteins containing TFA domains that might modulate transcription in a promoter context-dependent manner. TCEAL1, a TCEAL family member, is reported to be a nuclear phosphoprotein functioning to repress Rous sarcoma virus long-terminal repeat transcription and to contribute to preventing the cellular transformation induced by the Rous sarcoma virus long-terminal repeat\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. On the other hand, the closely related protein transcription elongation factor SII (TFIIS/TCEA1) has been reported to be implicated in transcription elongation and transcript fidelity\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe TCEAL gene family is implicated in various biological processes and diseases. TCEAL1 is associated with X-linked dominant neurodevelopmental syndromes and influences neurological traits\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. TCEAL2 has been highlighted as a prognostic marker in pan-cancer studies, indicating its significance in cancer biology\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. TCEAL2 has also been reported to function as a tumor suppressor in renal cell carcinoma and to correlate with better patient prognosis\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Furthermore, TCEAL7 is recognized as a potential tumor suppressor gene, with a particular role in negatively regulating the NF-kappaB pathway, and is involved in the regulation of c-Myc activity in alternative lengthening of telomeres\u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The diverse functions of the TCEAL gene family members underline their importance in both development and disease.\u003c/p\u003e \u003cp\u003eCompared to other TCEAL family members, TCEAL5 remains a relatively understudied member of the TCEAL gene family. While scarce, emerging evidence suggests TCEAL5's roles in cellular proliferation, cardiac health, and muscle cell differentiation\u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In the present study, we report that TCEAL5 expression is decreased in glioma and forced expression of TCEAL5 inhibits cell migration and invasion. Mechanistic study demonstrates that TCEAL5 interacts with the NuRD complex to repress mesenchymal marker genes expression, thus to inhibit epithelial-mesenchymal transition (EMT) in glioma. These results indicate that TCEAL5 may function as a tumor repressor in human glioma.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Data source and statistical analysis\u003c/h2\u003e \u003cp\u003eData on mRNA expressions and the clinical traits of human glioma samples and normal samples were obtained from the TCGA (The Cancer Genome Atlas Program (TCGA)), CGGA (Chinese Glioma Genome Atlas) and GTEx (Genotype-Tissue Expression) initiatives. The mRNA expression datasets underwent processing via the UCSC Toil RNAseq Recompute approach, ensuring a unified analysis across various datasets devoid of batch effects (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://xenabrowser.net/hub/\u003c/span\u003e\u003cspan address=\"https://xenabrowser.net/hub/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Statistical evaluations were performed utilizing the R software, version 4.2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.r-project.org/\u003c/span\u003e\u003cspan address=\"http://www.r-project.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). To explore TCEAL5's clinical relevance in glioma, its expression levels were categorized into high and low groups, based on an optimal cutoff value determined by the surv_cutoff function of the survminer R package. The log-rank test was employed to compare survival curves across these groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cell lines and cell culture\u003c/h2\u003e \u003cp\u003eU251 cells and Ln229 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), which performs routine cell line authentication testing with short tandem repeat analysis. Mycoplasma contamination was regularly examined and no contamination occurred during this study. Cells were cultured as previously described \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. TCEAL5 cDNA was cloned into the GV492 lentivirus vector (GeneChem, China) to enable ectopic expression in human glioma cells. Lentiviruses were produced by the transfection of 293T cells with plasmids using the packaging Mix (GeneChem, China). For TCEAL5 ectopic expression, glioma cells were infected with the lentiviruses and selected with puromycin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 RNA extraction and reverse transcription-quantitative PCR (RT-qPCR)\u003c/h2\u003e \u003cp\u003eCells were subjected to total RNA extraction using Invitrogen's Trizol reagent (Cat. No. 15596026), adhering strictly to the provided guidelines. Post-extraction, the integrity and concentration of the RNA were assessed through gel electrophoresis and measured using the Thermoscientific Nanodrop2000 spectrophotometer. To eliminate genomic DNA contamination and synthesize cDNA, 2 \u0026micro;g of the RNA underwent reverse transcription utilizing Vazyme's HiScript III RT SuperMix for qPCR (+\u0026thinsp;g DNA wiper) (Cat. No. R123-01), in compliance with the supplier's protocol. The synthesized cDNA was then analyzed using quantitative real-time PCR (qPCR) on the ABI-7500 system, employing TAKARA's TB Green\u0026trade; Premix Ex Taq\u0026trade; (Tli RNaseH Plus) (Cat. No. RR420A). β-actin was employed as the internal control for normalization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Western blot and antibodies\u003c/h2\u003e \u003cp\u003eThe cells were processed for protein extraction by lysing them in RIPA (150 mM NaCl, 2 mM EDTA, 50 mM Tris pH 8.0, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail). Post-lysis, the lysate was centrifuged at 12,000 g and 4\u0026deg;C for 20 minutes. The protein content of the supernatant was then quantified using the Bradford protein assay. The whole cell extracts were subjected to SDS-PAGE, and were subsequently transferred onto a PVDF membrane. The membrane was then blocked using a 5% solution of nonfat dry milk in TBST to prevent non-specific binding. This was followed by incubation with primary antibodies overnight at 4\u0026deg;C, and then with HRP-conjugated secondary antibodies at room temperature for 2 hours. Finally, the chemiluminescence reaction was carried out according to the manufacturer's instructions. The primary antibodies used in Western blot are as follows: anti-FLAG (Sigma, F3165), anti-E-cadherin (Proteintech, 20874-1-AP), anti-N-cadherin (Proteintech, 22018-1-AP), anti-TWIST1 (Proteintech, 25465-1-AP), anti-MTA2 (Proteintech, 17554-1-AP), anti-HDAC1 (Proteintech, 10197-1-AP), anti-CHD4 (Proteintech, 14173-1-AP), anti-β-tubulin (Proteintech, 66240-1-Ig).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Immunofluorescence and antibodies\u003c/h2\u003e \u003cp\u003eCells, cultivated on glass coverslips placed in 24-well plates, underwent fixation using a 4% solution of formaldehyde and were then made permeable with a PBS solution infused with 0.2% Triton X-100. A blocking step was conducted using PBS mixed with 5% bovine serum albumin for one hour, followed by a room temperature incubation with the primary antibody for another hour. This was succeeded by a one-hour incubation with secondary antibodies conjugated to a fluorescent dye, and subsequent DAPI staining. Microscopic imaging of the cells was carried out using a Leica microscope setup. The following antibodies were used in immunofluorescence: anti-FLAG (Sigma, F3165), anti-E-cadherin (Proteintech, 20874-1-AP), anti-N-cadherin (Proteintech, 22018-1-AP), anti-TWIST1 (Proteintech, 25465-1-AP).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Wound healing assays and transwell cell invasion assays\u003c/h2\u003e \u003cp\u003eIn the wound-healing assay, cells were cultured in 6-well plates. An overnight serum starvation was initiated, post which a sterile 1ml pipette tip was used to create a scratch, simulating a wound in the cell monolayer. After removing the dislodged cells with a PBS rinse, the culture was continued in a medium enriched with 1% FBS. The healing process, particularly the closure of the gap, was monitored and documented at specified time points using a light microscope.\u003c/p\u003e \u003cp\u003eFor the transwell in vitro cell invasion assays, cells, approximately 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e, were placed in the Matrigel pre-coated top chamber of the transwell chambers (8.0 mm, Corning), while the lower wells received complete medium to act as a chemoattractant and encourage cell invasion. The setup was then incubated for a period ranging from 24 to 48 hours within a cell culture incubator. Following incubation, cells that remained on the upper side of the membrane were carefully wiped away with a cotton swab. The cells that successfully migrated and adhered to the bottom side of the membrane were then stained with 0.1% Crystal Violet. The quantification of these cells was carried out manually to assess the invasion capability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Cell proliferation assay and clonogenic assay\u003c/h2\u003e \u003cp\u003eCell proliferation was assessed employing the Cell Counting Kit-8 (Beyotime, C0038), strictly in line with the instructions provided by the manufacturer. The procedure involved seeding cells at a density of 3\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells per well in 96-well plates. After the seeding, 100\u0026micro;l of serum-free culture medium, supplemented with 10\u0026micro;l of WST-8 reagent, was introduced to the cells at pre-determined time intervals. The plates were then placed in a cell culture incubator and allowed to incubate for 2 hours. Post-incubation, the optical absorbance of each well was measured at a wavelength of 450nm using a microplate reader to evaluate cell proliferation.\u003c/p\u003e \u003cp\u003eIn parallel, clonogenic assays were executed to examine the colony-forming ability of the cells. This involved seeding cells in 6-well plates at a density of 1000 cells per well. The seeded cells were incubated in a cell culture incubator for a duration of 14 days, allowing colonies to form. At the end of the incubation period, cells were stained using crystal violet staining solution (Beyotime, C0121) and the colonies were counted manually to quantify the clonogenic potential of the cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Immunoprecipitation\u003c/h2\u003e \u003cp\u003eTo conduct co-immunoprecipitation experiments, cells were lyzed using lysis buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, protease inhibitor cocktail) for 20 minutes at 4\u0026deg;C. Subsequent centrifugation at 14,000 g for 15 minutes at 4\u0026deg;C followed. The resulting protein supernatant was then incubated with 2 \u0026micro;g of specific antibodies for 12 hours at 4\u0026deg;C under constant rotation. Afterwards, 50 \u0026micro;l of a 50% solution of protein A or G agarose beads was added, extending the incubation for an additional 2 hours. The beads were washed five times with the original lysis buffer, with centrifugation at 500 g for 3 minutes at 4\u0026deg;C to collect the beads between washes. Proteins bound to the beads were eluted by resuspending in 2x SDS PAGE loading buffer and heated for 5 minutes. To identify the TCEAL5-interacting proteins, the eluates were then separated on a SDS-PAGE gel, silver stained, and analyzed through mass spectrometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Chromatin immunoprecipitation\u003c/h2\u003e \u003cp\u003eCells underwent crosslinking with 1% formaldehyde for 10 minutes at room temperature, which was then quenched by adding glycine to achieve a final concentration of 125 mM for 5 minutes. Subsequently, the cells were prepared in lysis buffer (1% SDS, 5 mM EDTA, and 50 mM Tris-HCl pH 8.0, protease inhibitors). This mixture was then exposed to cycles of sonication for 30 seconds on and off using a Bioruptor, resulting in chromatin fragments approximately 300 bp long. The lysates were then diluted in Dilution Buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl pH 8.0, protease inhibitors). For immunoprecipitation (IP), this diluted chromatin was incubated either with normal IgG as a control or with specific antibodies for 12 hours at 4\u0026deg;C under continuous rotation. Following this, 50 \u0026micro;L of 50% (volume/volume) protein A/G Sepharose beads were added, and the incubation continued for an additional 2 hours. The beads were then washed using wash buffers sequentially: Wash buffer I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, and 20 mM Tris-HCl, pH 8.0); Wash buffer II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, and 20 mM Tris-HCl, pH 8.0); Wash buffer III (0.25 M LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA, and 10 mM Tris-HCl, pH 8.0); and TE (1 mM EDTA and 10 mM Tris-HCl, pH 8.0). Finally, the chromatin complexes and input were de-crosslinked at 55\u0026deg;C for 12 hours in elution buffer (1% SDS and 0.1 M NaHCO3), and the DNA was purified using the QIAquick PCR Purification Kit. The DNA was then analyzed using quantitative real-time PCR (qPCR) on the ABI-7500 system, employing TAKARA's TB Green\u0026trade; Premix Ex Taq\u0026trade; (Tli RNaseH Plus) (Cat. No. RR420A).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1 TCEAL5 expression is significantly decreased in human glioma and higher expression favors prognosis\u003c/h2\u003e \u003cp\u003eTo explore TCEAL5's role in human glioma, an initial examination of its expression levels was conducted by analyzing RNA-sequencing data from both the TCGA (The Cancer Genome Atlas) and GTEx (Genotype-Tissue Expression) datasets. The analysis revealed a significant decrease in TCEAL5 expression in glioma samples compared to normal tissue samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To further deepen the investigation, a survey of TCEAL5 expression levels across histologic grades was carried out. The results indicated a negative correlation between TCEAL5 expression and histologic grades, suggesting a potential inverse relationship between TCEAL5 expression and malignancy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). These findings collectively point towards the possible significance of TCEAL5 in the progression of glioma.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further explore TCEAL5's clinical relevance in glioma, its expression levels were categorized into high and low groups, based on an optimal cutoff value determined by the surv_cutoff function of the survminer R package. The log-rank test was employed to compare survival curves across these groups. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, a notable association was observed between higher TCEAL5 expression and improved overall survival rates in glioma patients, underscoring a potential prognostic significance of TCEAL5 expression. This association was further supported by analysis using datasets from the CGGA, reinforcing the observation that higher TCEAL5 expression might be indicative of a more favorable prognosis in glioma (Fig. D). Collectively, these findings suggest a pivotal role for TCEAL5 in the pathology of glioma and its potential as a biomarker for patient prognosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2 TCEAL5 inhibits glioma cell migration and invasion\u003c/h2\u003e \u003cp\u003eTo investigate TCEAL5's regulatory influence on glioma, TCEAL5 was ectopically expressed in two glioma cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). This intervention's impact on cell dynamics was assessed through Cell Counting Kit 8 (CCK8) analysis for cell proliferation and a colony formation assay to gauge the cells' capacity for clone formation, a potential indicator of tumor growth potential. As depicted in Supplementary Figure S1, the overexpression of TCEAL5 only led to minor alterations in cell proliferation and colony-forming ability in both cell lines. This outcome suggests TCEAL5's role in glioma cell biology likely involves other mechanisms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further explore TCEAL5's role in glioma, the focus shifted to its influence on cell migration and invasion, critical factors in cancer metastasis. The wound healing assay revealed that the overexpression of TCEAL5 notably impeded the movement of glioma cells, as evidenced in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC. This inhibitory effect on migration was further substantiated through a transwell cell invasion assay, which similarly demonstrated a marked reduction in the invasion capabilities of glioma cells upon TCEAL5 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Collectively, these findings underscore TCEAL5's role as a deterrent in the processes of glioma cell migration and invasion, highlighting its potential significance in mitigating the metastatic progression of the cancer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3 TCEAL5 regulates EMT genes in glioma\u003c/h2\u003e \u003cp\u003eBased on the observation that TCEAL5 hinders glioma cell migration and invasion, it was hypothesized that TCEAL5 might execute this function by repressing epithelial-mesenchymal transition (EMT) in glioma. EMT is a critical process through which cancer cells acquire the ability to migrate and invade, often culminating in metastasis. To examine the impact of TCEAL5 overexpression on EMT gene expression, both western blot assays and immunofluorescence staining assays were employed. The findings indicated a decrease in mesenchymal gene expression alongside an increase in epithelial gene expression upon TCEAL5 overexpression, suggesting that TCEAL5 overexpression induces a mesenchymal-to-epithelial transition (MET)-like process in glioma cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB; Supplementary Fig. S2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo validate the direct regulatory influence of TCEAL5 on EMT gene expression, chromatin immunoprecipitation assays were conducted. These assays confirmed the binding of TCEAL5 to the promoters of mesenchymal genes N-cadherin and TWIST1, while no TCEAL5 binding was observed at the promoter of the epithelial gene E-cadherin, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC. This binding illustrates a direct regulatory mechanism of TCEAL5 on N-cadherin and TWIST1. In contrast, TCEAL5's regulation of E-cadherin likely involves indirect mechanisms. These findings collectively underscore the critical role of TCEAL5 in the regulation of EMT and potentially in the metastatic progression of glioma.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4 TCEAL5 interacts with the NuRD complex in glioma cells\u003c/h2\u003e \u003cp\u003eIn an effort to decode the specific mechanisms through which TCEAL5 influences the expression of mesenchymal genes N-cadherin and TWIST1, co-immunoprecipitation assay was performed. This assay aimed to purify TCEAL5 and its interacting partners in glioma cells. Post-purification, the immunoprecipitation (IP) fractions underwent separation via SDS-PAGE, and the distinct bands observed in the gels were further analyzed using Mass spectrometry. The result showed that TCEAL5 was co-purified with multiple components of the NuRD complex, a chromatin remodeling complex, as depicted in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB. The validity of this interaction was reinforced through reciprocal co-immunoprecipitation assays, the results of which are displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC. This collection of evidence robustly suggests that TCEAL5 collaborates with the NuRD complex in glioma cells, pointing towards an intriguing regulatory relationship in the modulation of gene expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5 TCEAL5-mediated recruitment of the NuRD complex silences mesenchymal gene transcription\u003c/h2\u003e \u003cp\u003eNext, the investigation delved into whether TCEAL5's suppression of mesenchymal gene expression, specifically N-cadherin and TWIST1, is facilitated by the NuRD complex. Chromatin immunoprecipitation assays were utilized to assess the impact of TCEAL5 overexpression on the NuRD complex's presence at the promoters of these genes. The findings indicated an increase in the NuRD complex's occupancy at the N-cadherin and TWIST1 promoters, suggesting a recruitment mechanism by TCEAL5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGiven the NuRD complex's known association with transcriptional repression, largely due to the presence of HDAC1, the study further examined the potential consequences of this increased occupancy on histone acetylation at these promoters. The results revealed a reduction in the levels of H3K9ac and H3K27ac, histone marks typically associated with active transcription, upon TCEAL5 overexpression and corresponding increase in NuRD occupancy (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). This suggests a direct epigenetic modulation by TCEAL5, leading to the transcriptional repression of these mesenchymal genes.\u003c/p\u003e \u003cp\u003eTo solidify the understanding of TCEAL5's role at the transcriptional level, qRT-PCR was conducted to measure the mRNA levels of N-cadherin and TWIST1 following TCEAL5 overexpression. The results from this analysis aligned with the previous findings at the protein level, confirming that TCEAL5 indeed exerts an inhibitory influence on the transcription of N-cadherin and TWIST1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). These findings collectively underscore the intricate regulatory role of TCEAL5 in glioma, particularly in its ability to modulate the expression of crucial genes involved in cell migration and invasion through epigenetic mechanisms.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eTCEAL5 is a relatively understudied TCEAL gene family member. This study revealed that TCEAL5 expression is substantially decreased in human glioma, and this reduction is inversely correlated with the tumor malignancy, and higher TCEAL5 expression is associated with improved prognosis in glioma patients, underscoring its potential as a prognostic biomarker. Interestingly, despite the observed decline in TCEAL5 expression in glioma, its forced overexpression did not markedly alter cell proliferation or colony formation capabilities. This suggests that the influence of TCEAL5 extends beyond mere modulation of cell growth, a hypothesis substantiated by the subsequent observations of its role in impeding cell migration and invasion. These processes are critical in cancer metastasis, and the study's findings that TCEAL5 overexpression leads to reduced migration and invasion capabilities in glioma cells point to its potential role in mitigating metastatic progression.The mechanisms underlying TCEAL5's regulatory effects appear to involve the modulation of EMT gene expression. Our results demonstrated that TCEAL5 overexpression induces a MET-like process. The direct binding of TCEAL5 to the promoters of mesenchymal genes like N-cadherin and TWIST1, coupled with the indirect influence on the epithelial gene E-cadherin, reveals a nuanced regulatory mechanism through which TCEAL5 exerts its function.\u003c/p\u003e \u003cp\u003eAn intriguing aspect of TCEAL5's mechanism of action is its interaction with the NuRD complex, a chromatin remodeling complex known to influence gene expression. The study's findings suggest that TCEAL5 recruits the NuRD complex to the promoters of mesenchymal genes, leading to a reduction in histone acetylation and subsequent transcriptional repression of these genes. This epigenetic modulation offers a layer of complexity to the understanding of TCEAL5's role in glioma and underscores the potential for targeting these interactions in therapeutic interventions.\u003c/p\u003e \u003cp\u003eIn conclusion, this study sheds light on the pivotal role of TCEAL5 in glioma, presenting a comprehensive overview of its expression dynamics, regulatory mechanisms, and clinical implications. The insights gained pave the way for future research and potential therapeutic strategies, emphasizing the importance of TCEAL5 in the pathology of glioma and possibly other cancers.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTCEAL5\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etranscription elongation factor A like 5\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNuRD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNucleosome Remodeling Deacetylase complex\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEMT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEpithelial-Mesenchymal Transition\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTCGA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThe Cancer Genome Atlas\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCGGA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eChinese Glioma Genome Atlas.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all the members in Dr. Zhang\u0026rsquo;s lab for insightful scientific discussion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (81972653).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e―Approval of the research protocol by an Institutional Reviewer Board: N/A.\u003c/p\u003e\n\u003cp\u003e―Informed Consent: N/A.\u003c/p\u003e\n\u003cp\u003e―Registry and the Registration No. of the study/trial: N/A.\u003c/p\u003e\n\u003cp\u003e―Animal Studies: N/A.\u003c/p\u003e\n\u003cp\u003e―Author Contribution: Conceptualization: DZ; methodology: DZ, HZ, XL, YZ, QZ, XZ; validation: DZ, HZ, XL, YZ, QZ, XZ; investigation: DZ, HZ, XL, YZ, QZ, XZ; data curation: DZ, HZ; writing: DZ; visualization: DZ, HZ; supervision: DZ; funding acquisition: DZ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary materials files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYeh CH, Shatkin AJ (1995) A cis-acting element in Rous sarcoma virus long terminal repeat required for promoter repression by HeLa nuclear protein p21. J Biol Chem 270:15815\u0026ndash;15820\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJeon C, Agarwal K (1996) Fidelity of RNA polymerase II transcription controlled by elongation factor TFIIS. Proc Natl Acad Sci U S A 93:13677\u0026ndash;13682\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThomas MJ, Platas AA, Hawley DK (1998) Transcriptional fidelity and proofreading by RNA polymerase II. Cell 93:627\u0026ndash;637\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHijazi H, Reis LM, Pehlivan D et al (2022) TCEAL1 loss-of-function results in an X-linked dominant neurodevelopmental syndrome and drives the neurological disease trait in Xq22.2 deletions. Am J Hum Genet 109:2270\u0026ndash;2282\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun Y, Zhao J (2022) Transcription Elongation Factor A (SII)-Like (TCEAL) Gene Family Member-TCEAL2: A Novel Prognostic Marker in Pan-Cancer. Cancer Inform 21:11769351221126285\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou Y, Zhang Y, Li W et al (2020) TCEAL2 as a Tumor Suppressor in Renal Cell Carcinoma is Associated with the Good Prognosis of Patients. Cancer Manage Res 12:9589\u0026ndash;9597\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRattan R, Narita K, Chien J et al (2010) TCEAL7, a putative tumor suppressor gene, negatively regulates NF-kappaB pathway. Oncogene 29:1362\u0026ndash;1373\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrhan C, Bulut P, Dalay N, Ersen E, Buyru N (2019) Downregulation of TCEAL7 expression induces CCND1 expression in non-small cell lung cancer. Mol Biol Rep 46:5251\u0026ndash;5256\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLafferty-Whyte K, Bilsland A, Hoare SF et al (2010) TCEAL7 inhibition of c-Myc activity in alternative lengthening of telomeres regulates hTERT expression. Neoplasia 12:405\u0026ndash;414\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSawada A, Yamamoto T, Sato T (2022) Tceal5 and Tceal7 Function in C2C12 Myogenic Differentiation via Exosomes in Fetal Bovine Serum. Int J Mol Sci. ; 23\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao X, Hu D, Li J, Zhao G, Tang W, Cheng H (2020) Database Mining of Genes of Prognostic Value for the Prostate Adenocarcinoma Microenvironment Using the Cancer Gene Atlas. \u003cem\u003eBioMed research international\u003c/em\u003e. ; 2020: 5019793\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWan G, Chen P, Sun X et al (2021) Weighted gene co-expression network-based approach to identify key genes associated with anthracycline-induced cardiotoxicity and construction of miRNA-transcription factor-gene regulatory network. Mol Med 27:142\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGergics P, Christian HC, Choo MS, Ajmal A, Camper SA (2016) Gene Expression in Mouse Thyrotrope Adenoma: Transcription Elongation Factor Stimulates Proliferation. Endocrinology 157:3631\u0026ndash;3646\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShao S, Cao H, Wang Z et al (2020) CHD4/NuRD complex regulates complement gene expression and correlates with CD8 T cell infiltration in human hepatocellular carcinoma. Clin epigenetics 12:31\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"TCEAL5, NuRD, EMT, Glioma, Tumor suppressor","lastPublishedDoi":"10.21203/rs.3.rs-3907845/v3","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3907845/v3","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe TCEAL5 gene, a member of the TCEAL family, is linked to various biological processes but remains understudied in cancer research. This study analyzed TCEAL5 expression in glioma and investigated its biological functions through cell assays and molecular analyses. Our findings revealed a significant reduction in TCEAL5 expression in glioma tissues, with lower expression levels correlating with higher histologic grades and poorer prognosis. Further experimental investigations demonstrated that ectopic overexpression of TCEAL5 in glioma cell lines significantly inhibited cell migration and invasion. Mechanistic studies indicated that TCEAL5 exerts its inhibitory effects on EMT by directly binding to the promoters of mesenchymal genes. Additionally, TCEAL5 was found to interact with the NuRD complex, leading to transcriptional repression of mesenchymal genes via epigenetic modulation. These findings highlight the multifaceted role of TCEAL5 as a tumor suppressor in glioma, suggesting its potential as a prognostic biomarker and a target for therapeutic intervention. Our study not only adds to the understanding of TCEAL5's biological functions but also opens new avenues for research into its application in cancer therapy.\u003c/p\u003e","manuscriptTitle":"TCEAL5 cooperates with the NuRD complex to epigenetically silence mesenchymal genes in glioma","msid":"","msnumber":"","nonDraftVersions":[{"code":3,"date":"2024-04-12 20:10:52","doi":"10.21203/rs.3.rs-3907845/v3","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":2,"date":"2024-04-11 15:17:32","doi":"10.21203/rs.3.rs-3907845/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2024-02-01 16:12:50","doi":"10.21203/rs.3.rs-3907845/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7aa71b39-8661-4b9a-b3cb-ae2d9f98e28d","owner":[],"postedDate":"April 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-18T09:57:16+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-12 20:10:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v3","identity":"rs-3907845","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3907845","identity":"rs-3907845","version":["v3"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}