UBE2T promotes PTC progression by activating the JAK/STAT3 pathway via negative regulation of SOCS2

UBE2T promotes PTC progression by activating the JAK/STAT3 pathway via negative regulation of SOCS2 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article UBE2T promotes PTC progression by activating the JAK/STAT3 pathway via negative regulation of SOCS2 Lijun Zhang, Chengyuan Li, Jianing Zhou, Lin Li, Xiang Zhang, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5649270/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Thyroid cancer, particularly papillary thyroid carcinoma (PTC), is one of the most common malignant tumors of the endocrine system. Malignant biological behaviors such as tumor invasion and cervical lymph node metastasis are closely associated with the prognosis of PTC. To date, no effective method has been identified to accurately predict the invasive biological behavior of PTC. Objective This study aims to investigate the potential molecular mechanisms underlying the high invasiveness of PTC mediated by UBE2T. Methods We examined the expression of UBE2T in PTC using data from the TCGA and GEO databases and validated these findings in clinical samples from our institution, analyzing clinical pathological features. Subsequently, we explored the impact of UBE2T on the biological behavior of PTC cells through stable overexpression or knockdown of the UBE2T gene. Additionally, we elucidated the potential mechanisms by which UBE2T promotes PTC progression, with a particular focus on its role in activating the JAK-STAT signaling pathway. Results Our results demonstrate that UBE2T plays a crucial role in promoting PTC progression by activating the JAK-STAT signaling pathway. Correlation analysis and co-immunoprecipitation (co-IP) experiments identified cytokine signaling suppressor 2 (SOCS2) as a key molecule mediating UBE2T's action in the JAK-STAT pathway. Further rescue experiments and immunofluorescence (IF) assays confirmed that UBE2T promotes PTC progression by negatively regulating SOCS2, thereby activating the JAK-STAT3 pathway. Conclusion This study reveals the mechanistic role of UBE2T in the high invasiveness of PTC, highlighting its negative regulation of SOCS2 to activate the JAK-STAT3 signaling pathway and drive PTC progression. These findings provide new insights into the mechanisms of PTC invasion and may offer potential therapeutic targets for inhibiting PTC metastasis and recurrence. UBE2T thyroid carcinoma SOCS2 JAK/STAT3 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Thyroid cancer is the most prevalent endocrine malignancy, with a dramatic rise in incidence over the past two decades, making it the eighth most common malignancy globally and the fourth most frequent cancer among women[ 1 , 2 ]. This surge is primarily attributed to the increasing prevalence of PTC, which accounts for the majority of thyroid cancer cases. While most PTCs exhibit indolent growth and favorable prognosis, a subset (10–15%) demonstrates aggressive phenotypes characterized by extrathyroidal invasion and cervical lymph node metastasis, which are strongly associated with poor clinical outcomes. Despite extensive efforts to elucidate the molecular underpinnings of PTC aggressiveness, particularly through studies on pathways involving BRAF, TERT, and MAPK[ 3 , 4 ], effective methods for predicting or mitigating its invasive behavior remain elusive. UBE2T, a member of the E2 enzyme family, has emerged as a critical oncogenic player in multiple malignancies, such as cholangiocarcinoma[ 5 ], colorectal[ 6 ], esophageal[ 7 ], lung[ 8 ], and pancreatic cancers[ 9 ]. By promoting proliferation, migration, and invasion through diverse molecular pathways, UBE2T has been highlighted as a potential therapeutic target. Our preliminary findings revealed that UBE2T is significantly upregulated in PTC tissues compared to normal thyroid tissues, correlating with aggressive features such as lymph node metastasis and extrathyroidal invasion. However, the molecular mechanisms underlying UBE2T-driven PTC progression remain poorly defined. The development of tumors is often driven by signaling dysregulation resulting from gene mutations or abnormal gene expression. These aberrantly activated signaling pathways are core mechanisms that drive cell proliferation, immune evasion, anti-apoptosis, and invasion/metastasis[ 10 – 12 ]. For example, pathways such as MAPK, PI3K/Akt, and JAK/STAT, when aberrantly activated, can continuously transmit "growth" signals, enabling tumor cells to escape normal regulation. In PTC, the most commonly dysregulated pathway is the MAPK signaling pathway[ 13 ]. Approximately 70%-80% of PTC cases exhibit BRAF V600E mutations and RAS gene mutations, leading to sustained activation of MEK and ERK, which promotes tumor progression. It remains an open question whether UBE2T also promotes tumor progression in PTC through modulation of these classical signaling pathways. This is a key issue that warrants further investigation. In this study, we aimed to elucidate the molecular mechanisms by which UBE2T mediates PTC aggressiveness. Using TCGA and GEO databases, along with clinical validation, we analyzed UBE2T expression and its association with clinicopathological features in PTC. Functional assays were conducted to assess the effects of UBE2T overexpression and knockdown on PTC cell behaviors. Mechanistically, we demonstrated that UBE2T promotes PTC progression by activating the JAK/STAT3 signaling pathway through the suppression of SOCS2, a key inhibitory molecule in the pathway. These findings were further validated through co-IP, IF, and rescue experiments, highlighting UBE2T as a potential therapeutic target in aggressive PTC. Materials and methods Public data We downloaded the uniformly standardized pan-cancer dataset from the UCSC ( https://xenabrowser.net/ ) database, which includes TCGA, TARGET, and GTEx. We filtered out samples with an expression level of 0 and excluded cancer types with fewer than three samples within a single cancer group. Ultimately, expression data from 34 cancer types were obtained for single-gene pan-cancer analysis. Additionally, we retrieved the mRNA expression matrix file for TCGA-THCA from the Genomic Data Sharing (GDS) portal ( https://www.ncbi.nlm.nih.gov/ ) and the mRNA expression matrix file for normal thyroid tissue from GTEx for subsequent correlation analysis. Clinical Samples This study utilized 100 pairs of tissue samples collected from patients who underwent surgery at Jiangsu Provincial People's Hospital (The First Affiliated Hospital of Nanjing Medical University) between January 2022 and December 2023. The study was approved by the hospital's ethics committee (Ethics Approval No. 2023-SR-425), and all participants provided written informed consent. Quantitative RT–PCR Add 0.5-1 mL of Trizol reagent to an appropriate amount of tissue or culture dish, followed by the addition of 1/5 the volume of chloroform. Incubate for 10 minutes and centrifuge at 12,000g for 15 minutes at 4°C. Transfer the upper aqueous phase to a new tube, add an equal volume of isopropanol, shake, and incubate for 10 minutes. Centrifuge under the same conditions, discard the supernatant, and retain the pellet. Add 1 mL of 75% ethanol, centrifuge at 12,000g for 10 minutes, and repeat the wash once. Retain the pellet to obtain total RNA. The RNA concentration was measured using a Nanodrop (Thermo Fisher). RNA was reverse transcribed into cDNA using the FastKing gDNA Dispelling RT SuperMix (TIANGEN, KR118-01) kit, with 500 ng of RNA used in each reverse transcription reaction. qPCR was performed using SuperReal PreMix Plus (SYBR Green) (TIANGEN, FP205), and data were collected and analyzed using the Applied Biosystems PCR System (StepOnePlus Real-Time PCR System, Thermo Fisher). Western Blot Western Blot Protein extraction from cells or tissue samples was performed under low-temperature conditions. An appropriate amount of Protease Inhibitor Cocktail (100X) (CST, 5871) was added to RIPA lysis buffer (Servicebio, G2002). The samples were sonicated at 30% power for 20 seconds, followed by a 20-second ice bath, repeated three times. The samples were then centrifuged at 15,000 rpm for 20 minutes at 4°C. The supernatant was collected, and protein concentration was determined using a BCA assay kit. Protein loading buffer (4:1 volume ratio) was added to the protein extract in a pre-chilled EP tube and thoroughly mixed. The samples were denatured by heating in a 100°C water bath for 10 minutes. Protein separation was performed using 7.5% (Epizyme Biotech, PG211) or 10% SDS-PAGE gel (Epizyme Biotech, PG212), and the proteins were transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk solution for 1.5 hours, followed by incubation with the corresponding primary antibody at 4°C overnight. On the following day, the membrane was washed three times with TBST, each wash for 10 minutes. The membrane was then incubated with the secondary antibody at room temperature for 2 hours, washed again with TBST three times, each wash for 10 minutes, and finally exposed. Immunohistochemistry (IHC) Paraffin-embedded tissue sections (4 µm) were baked at 65°C for 1 hour, deparaffinized in xylene, and rehydrated in a gradient of ethanol. Antigen retrieval was performed by microwaving the sections in pH 6.0 sodium citrate buffer for 10 minutes at high temperature, followed by cooling and washing the sections three times with PBS. The sections were incubated with 3% hydrogen peroxide solution at room temperature for 10 minutes to block endogenous peroxidase activity, and then blocked with 5% normal goat serum at room temperature for 30 minutes. The UBE2T antibody (Thermo Fisher, PA5-28464) was applied and incubated overnight at 4°C. The next day, after PBS washing, biotinylated secondary antibody was added and incubated at room temperature for 30 minutes. After another PBS wash, HRP-conjugated streptavidin was added and incubated for 30 minutes. DAB was used for color development for 2–5 minutes, and the reaction was terminated by rinsing with tap water. The sections were counterstained with hematoxylin for 1 minute, dehydrated through a gradient of ethanol, cleared in xylene, and mounted. The results were observed under a microscope. Lentiviral Infection The TPC-1 cells (FuHeng BioLogy, FH1039) and KTC-1 cells (Servicebio, STCC12507P) were adjusted to a concentration of 6×104 cells/mL and seeded at 2 mL per well in a 6-well plate. The infection was performed when the cell density reached 80%-90% the following day. Before infection, lentivirus suspension (purchased from Genechem) was prepared, and the required viral amount was calculated based on the MOI. The viral suspension was mixed with complete medium, gently mixed, and added to the cell culture plate.The cells were incubated at 37°C in a 5% CO 2 incubator for 8–12 hours. After the incubation, the fresh complete medium was replaced, and the cells continued to be cultured. After 48–72 hours of infection, puromycin was used to select positive cell clones. Plasmid Transfection Before the experiment, PTC cells were seeded in 6-well plates at 60%-80% confluence to ensure they were in a good growth condition. 0.5-2 µg of plasmid DNA (purchased from Genechem) was added per well. The plasmid DNA was diluted in serum-free medium and mixed with the transfection reagent Lipo3000 (Biosharp, BL632A). The plasmid DNA and transfection reagent were gently mixed and allowed to stand at room temperature for 15–20 minutes to form a DNA-transfection reagent complex.The original culture medium was removed, and serum-free medium was added. The DNA-transfection reagent complex was then added to the cells, gently mixed to ensure uniform distribution, and the cells were incubated in a 37°C, 5% CO 2 incubator for 4–6 hours. Afterward, the medium was replaced with complete medium containing serum, and the cells were further cultured. Colony Formation Assay Cells were seeded at a density of 2×10 5 cells per well in a 6-well plate. After the cells adhered, they were transfected with plasmids. After 48 hours, cells in the logarithmic growth phase were collected, counted using a cell counting chamber, and approximately 400 cells were seeded per well. The cells were then cultured in a 37°C, 5% CO 2 incubator for 14 days, with medium changes as needed. After 14 days, cells were fixed with 3% paraformaldehyde and stained with crystal violet. The colonies were photographed and counted. CCK-8 Assay Cells were seeded at 2×10 3 cells per well in 100 µL medium in a 96-well plate. After 24 hours of incubation at 37°C with 5% CO 2 , 10 µL of CCK-8 solution (ApexBio, K1018) was added to each well. The cells were incubated in the incubator for 1.5 hours, and the absorbance at 450 nm was measured. Wound Healing Assay Three horizontal lines were drawn evenly on the bottom of a 6-well plate. After seeding 2×10 5 cells per well and allowing them to adhere, cells were transfected with plasmids. After 48 hours, a 20 µL pipette tip was used to create two vertical lines in each well. PBS was used to wash away floating cells, and wound healing was observed for 12–48 hours. A camera was used to capture images of the wound healing process. Transwell Assay Cells were seeded at a density of 2×10 5 cells per well in a 6-well plate. After allowing the cells to adhere, they were transfected with plasmids. After 48 hours, cells in the logarithmic growth phase were collected, counted using a cell counting chamber, and resuspended in serum-free medium. The cell concentration was adjusted to 10^5 cells/mL and mixed thoroughly.For the invasion assay, Matrigel was added to the upper chamber of the Transwell. In each well, 500 µL of complete medium was added to the lower chamber, and 200 µL of the cell suspension was added to the upper chamber. The plate was incubated at 37°C in a cell culture incubator, with TPC-1 cells incubated for 24 hours and KTC-1 cells for 18 hours. After incubation, the chamber was removed, washed twice with PBS, fixed with 4% paraformaldehyde for 15 minutes, and stained with 0.1% crystal violet for 20 minutes. Unbound crystal violet and non-migrated cells on the upper side of the chamber were removed. Finally, the cells were observed and counted under a microscope. Co-IP Assay UBE2T-overexpressing or UBE2T-knockdown cells were lysed in IP lysis buffer (Thermo Fisher, 87787) and incubated on ice for 30 minutes. After centrifugation at 12,000 rpm for 15 minutes at 4°C, the cell lysate was incubated with primary antibodies against SOCS2 (Abcam, ab109245) and Flag (Beyotime, AF519) at 4°C overnight. The immunocomplex was then incubated with Dynabeads magnetic beads (Thermo Fisher, 10003D) for 4 hours at 4°C. After washing the immunocomplex three times, protein sample buffer was added, and the samples were boiled for denaturation before performing WB analysis. Animal Experiments 4-6-week-old BALB/c nude mice (Charles River, CAnN.Cg- Foxn1 nu /Crl) were housed in an SPF environment. After culturing cells to the logarithmic growth phase (80–90% confluence), the cells were digested with trypsin to prepare a cell suspension (4×10 7 cells/mL), which was kept on ice. For subcutaneous implantation, 100µL of cell suspension was injected subcutaneously into the axillary region of nude mice, and tumor growth was monitored regularly. When tumor volume reached approximately 2000 mm³, samples were collected. For lung metastasis experiments, the cell suspension (1–2×10 6 cells/mL) was injected via the tail vein (100–200µL per mouse). After injection, the mice were kept in a controlled temperature and humidity environment, and tumor metastasis was monitored using fluorescence imaging over a 2–6 week period. All animal experiments were conducted in accordance with the guidelines of the Animal Ethics Committee of Nanjing Medical University. IF Staining: Paraffin-embedded tissue sections (4–6µm) were deparaffinized and rehydrated, followed by antigen retrieval with citrate buffer (pH 6.0) using high-pressure cooking. After blocking with 5% normal serum for 30 minutes, the sections were incubated with primary antibodies against UBE2T (GeneTex, GTX83452) and SOCS2 (Abcam, ab109245) in a humidity chamber at 4°C overnight. The next day, the sections were washed with PBS and incubated with corresponding fluorescent-labeled secondary antibodies at room temperature for 1 hour, protected from light. Nuclei were stained with DAPI, and after PBS washing, the sections were mounted with anti-fluorescence quenching mounting medium. Images were captured using a laser confocal microscope. The entire process was carried out under strict light protection to prevent fluorescence quenching. Statistical Analysis Expression differences between normal and tumor samples in each tumor were calculated using R software (version 3.6.4). Statistical significance was analyzed using the unpaired Wilcoxon Rank Sum and Signed Rank Tests. Student's t-test was used for statistical comparisons between two experimental conditions with unpaired samples, while the Wilcoxon Rank Sum test was used for all paired t-tests. One-way analysis of variance (ANOVA) was used for comparisons among multiple experimental groups. Disease-Free Interval (DFI) analysis was performed using Kaplan-Meier plots. *P < 0.05, **P < 0.01, and ***P < 0.001 indicate statistical significance. Results UBE2T is Overexpressed in PTC and Correlates with Aggressive Characteristics To explore the potential role of UBE2T in the development of PTC, we conducted a comprehensive analysis of UBE2T expression in tumor and corresponding normal tissues across various cancers using gene expression data from the TCGA, TARGET, and GTEx databases. The results indicated that UBE2T mRNA expression was significantly upregulated in 33 different tumor types (Figure 1A). Further differential analysis of RNA-seq data from 59 paired PTC samples in the TCGA database confirmed that UBE2T expression was significantly elevated in PTC tissues compared to normal tissues (Figure 1B). Subsequently, we performed qRT-PCR to assess UBE2T expression in 100 pairs of PTC and adjacent non-tumor tissues that met inclusion and exclusion criteria at our institution. The results showed that UBE2T expression was significantly higher in PTC tissues than in adjacent non-tumor tissues, with a statistically significant difference (Figure 1C). Western blotting analysis of UBE2T protein expression in 20 paired PTC and adjacent non-tumor tissues also revealed significantly higher protein levels in PTC tissues (Figure 1D). To further investigate the potential clinical and pathological effects of UBE2T, we examined UBE2T expression in human PTC samples by IHC. The IHC scores confirmed that UBE2T was more highly expressed in tumor tissues compared to adjacent non-tumor tissues (Figure 1E). Kaplan-Meier survival analysis showed that patients with low UBE2T expression had significantly longer DFI compared to those with high UBE2T expression (Figure 1F). Notably, higher UBE2T expression was closely associated with advanced tumor staging, lymph node metastasis, and extraglandular invasion (Figure 1G). UBE2T Promotes PTC Cell Proliferation and Migration To further investigate the regulatory role of UBE2T in PTC, we constructed stable UBE2T-overexpressing PTC cell lines (TPC-1 and KTC-1) using lentivirus. The transfection efficiency was evaluated by Western blot and RT-qPCR (Figure 2A, B). We also used plasmids to effectively knock down UBE2T expression (Figure 3A, B). As expected, in vitro experiments showed that overexpression of UBE2T promoted PTC cell proliferation compared to control cells (Figure 2C, D). In contrast, silencing UBE2T inhibited proliferation in TPC-1 and KTC-1 cells (Figure 3C, D). To assess the pro-migratory effect of UBE2T, we conducted wound healing and Transwell migration assays. Overexpression of UBE2T significantly enhanced the invasion and migration of TPC-1 and KTC-1 cells (Figure 2E, F), while UBE2T knockdown inhibited their invasion and migration (Figure 3E, F). Overall, these in vitro results suggest that UBE2T is a positive regulator of PTC cell proliferation and migration. UBE2T Activates the JAK1/STAT3 Signaling Pathway in PTC To further explore the mechanism by which UBE2T promotes thyroid cancer cell proliferation, invasion, and migration, we divided the samples into high (≥50% expression) and low (<50% expression) UBE2T expression groups and performed single-gene GSEA analysis. The results suggested that the JAK/STAT3 signaling pathway could be a key pathway through which UBE2T promotes PTC cell proliferation, invasion, and migration (Figure 4A, B). After overexpressing UBE2T in TPC-1 and KTC-1 cells, we measured the mRNA expression of JAK/STAT3 pathway target genes (BCL-2, CCND1, VEGFA) by qRT-PCR. The results showed that UBE2T overexpression upregulated the expression of these downstream target genes compared to controls (Figure 4C), while UBE2T knockdown significantly reduced the expression of BCL-2, CCND1, and VEGFA (Figure 4D). Western blotting of phosphorylated JAK1, JAK2, and STAT3 proteins showed significantly increased phosphorylation of JAK1 and STAT3 in UBE2T-overexpressing cells compared to controls (Figure 4E), suggesting that UBE2T may promote carcinogenesis through activation of the JAK1-STAT3 signaling pathway. To determine whether UBE2T activates this pathway by directly interacting with JAK1 or STAT3, we conducted Co-IP experiments. Unfortunately, the results did not support an interaction between UBE2T and these proteins (Figure S1A-B). To identify potential interacting molecules of UBE2T, we performed a correlation analysis using PTC samples from the TCGA-THCA dataset. The results indicated a significant negative correlation between UBE2T and SOCS2 expression (r = -0.42, p < 0.05) within the SOCS family (Figure 4F and Figure S1C-F). SOCS family members are important negative regulators of cytokine signaling, playing a role in feedback inhibition of the JAK-STAT pathway. Co-IP experiments confirmed that SOCS2 was successfully detected in UBE2T immunocomplexes, whereas no related signal was observed in the negative control (IgG group) (Figure 4G). This suggests that UBE2T interacts with SOCS2 to participate in relevant signal regulatory mechanisms. UBE2T Activates JAK1/STAT3 Pathway by Downregulating SOCS2 to Promote PTC Progression To further investigate whether UBE2T promotes PTC cell proliferation and invasion through its interaction with SOCS2, we performed rescue experiments. The results showed that overexpression of UBE2T promoted the proliferation, invasion, and migration of PTC cell lines, and upregulation of SOCS2 partially reversed these effects (Figure 5A-C). In contrast, knockdown of UBE2T inhibited the proliferation, invasion, and migration of TPC-1 and KTC-1 cells, and suppression of SOCS2 expression partially reversed these inhibitory effects (Figure S2A-C). Moreover, we found that overexpression of UBE2T upregulated the expression of downstream target genes in the JAK1/STAT3 pathway, and upregulation of SOCS2 partially reversed this effect (Figure 5D). Conversely, UBE2T knockdown reduced the expression of these target genes, and suppression of SOCS2 partially reversed this reduction (Figure S2D). To further confirm the pro-cancer role of UBE2T in PTC, we conducted in vivo experiments. The results showed that tumors from nude mice with upregulated UBE2T expression had significantly larger volumes and weights compared to controls (Figure 6A). In contrast, tumors from nude mice with UBE2T knockdown were significantly smaller than controls (Figure 6B). Next, we used a tail vein injection model to investigate the role of UBE2T in PTC metastasis in vivo. Fluorescence signal intensity measurements revealed that the lungs of mice overexpressing UBE2T exhibited significantly higher average fluorescence signal intensity compared to controls, with a statistically significant difference (Figure 6C). In contrast, mice with UBE2T knockdown showed lower fluorescence signal intensity in their lung tissues compared to controls (Figure 6D). These results suggest that UBE2T promotes PTC tumor proliferation and metastasis in vivo. To validate the relationship between UBE2T and SOCS2, we performed immunofluorescence staining on PTC tissues. The immunofluorescence results showed that UBE2T and SOCS2 predominantly co-localized in the cytoplasm (Figure 6E). This further supports that in PTC, UBE2T may influence tumor progression through its interaction with SOCS2. Discussion Our findings provide novel mechanistic insights into the oncogenic role of UBE2T in the progression of PTC. Through analysis of public datasets and institutional clinical samples, we demonstrated that UBE2T is significantly overexpressed in PTC tissues. Functional assays conducted both in vitro and in vivo revealed that UBE2T promotes malignant phenotypes, including enhanced cellular proliferation and invasion. Mechanistic investigations, incorporating GSEA and Western blot analyses, identified the JAK/STAT3 signaling pathway as a critical downstream effector mediating the tumor-promoting effects of UBE2T. The JAK/STAT3 pathway, an essential cellular signal transduction pathway, is widely involved in various cellular processes such as growth, differentiation, proliferation, apoptosis, and immune regulation[ 14 ]. In oncology research, the activation of the JAK/STAT3 pathway is frequently used as a prognostic marker. For example, overexpression of STAT3 is associated with poor prognosis in several solid tumors[ 15 – 17 ]. Additionally, STAT3 can enhance the immunosuppressive functions of tumor-associated macrophages (TAMs), inhibit CD8 + T cell activity, and promote the proliferation of regulatory T cells (Tregs), thereby aiding tumor cells in evading immune surveillance[ 16 ]. Given the pivotal role of the JAK/STAT3 pathway in tumor development, STAT3 has been considered an important therapeutic target, and several classic STAT3 inhibitors, such as Stattic, have been developed[ 18 ]. Moreover, strategies targeting the inhibition of STAT phosphorylation or blocking its DNA-binding have been explored. The development of STAT inhibitors and JAK kinase inhibitors has become a significant focus in recent cancer drug research. Our experimental findings show that UBE2T overexpression enhances the phosphorylation of JAK1 and STAT3, thereby activating this pathway. Furthermore, our study highlights the role of the oncogenic factor UBE2T in activating the JAK/STAT3 pathway through SOCS2. Previous studies have demonstrated that SOCS2 is a downstream molecule of JAK but exerts regulatory effects on the JAK signaling pathway through a negative feedback mechanism[ 19 , 20 ]. When cytokines such as IL-6, EPO, or GH activate JAK kinases through receptors, JAK phosphorylates specific sites on the receptor, recruiting and activating STAT proteins[ 21 ]. The activated STAT proteins enter the nucleus, bind to the promoter regions of target genes, and induce SOCS2 transcription. Once SOCS2 is produced, it negatively regulates JAK signaling by directly inhibiting JAK kinase activity or promoting the degradation of receptors or JAKs. SOCS2 has a dual role in various tumors, acting both as a tumor suppressor and, in certain contexts, promoting tumor progression[ 22 – 24 ]. In most tumors, including breast cancer[ 19 ], colorectal cancer[ 25 ] and pancreatic cancer[ 26 ], SOCS2 exerts a negative regulatory effect, which aligns with its tumor-suppressive role in PTC. Currently, there is limited research on the relationship between UBE2T, SOCS2, and JAK/STAT3 in the literature. Nevertheless, our results provide novel evidence emphasizing that UBE2T activates the JAK/STAT3 pathway by negatively regulating SOCS2, thus promoting the progression of PTC. This study has certain limitations, including the need for further clarification of the precise mechanism by which UBE2T regulates SOCS2, as well as the therapeutic potential of STAT3 inhibitors in blocking PTC progression. In conclusion, our study demonstrates the critical role of UBE2T in promoting the progression of PTC. UBE2T is highly expressed in PTC and activates the JAK/STAT3 pathway through SOCS2. These findings contribute to a deeper understanding of the molecular mechanisms underlying PTC progression and offer potential diagnostic and therapeutic targets for PTC invasiveness. Abbreviations Abbreviation Full Name PTC papillary thyroid carcinoma co-IP co-immunoprecipitation SOCS2 cytokine signaling suppressor 2 IF immunofluorescence GDS the Genomic Data Sharing IHC immunohistochemistry ANOVA one-way analysis of variance DFI disease-free interval TAMs tumor-associated macrophages Tregs regulatory T cells Declarations Funding Declaration This work was supported by Jiangsu Provincial Medical Key Discipline （No. ZDXK202222）. Ethics Approval and Consent to Participate This study was approved by the Ethics Committee of Jiangsu Province Hospital (Approval No. 2023-SR-425). All authors have consented to the publication of this manuscript in Cancer Cell International . Author Contribution Lijun Zhang: Conception and design of the study；acquisition and analysis of data, Drafting the manuscript and revising it and Ensure data integrity and support for transparency and reproducibility.Chengyuan Li: Acquisition of data from clinical samples and laboratory experiments, analysis and interpretation of results, including JAK-STAT3 pathway activation and Drafting sections of the manuscript and revising it.Jianing Zhou: Acquisition of clinical samples and data, Analysis and interpretation of clinical pathological features and Drafting sections of the manuscript and revising it.Lin Li: Design of experiments, Creation of new software (if applicable) for data analysis and Revising the manuscript and contributing to experimental design.Xiang Zhang: Data collection and analysis, Co-immunoprecipitation experiments and result interpretation and Revising the manuscript.Haisheng Fang: Conception and design of experiments, Interpretation of data related to the JAK-STAT3 signaling pathway and revising the manuscript.Jingsheng Cai: Acquisition of clinical data, Data analysis related to pathological features and Revising the manuscript.Houchao Tong: Conception and design of experiments focused on the JAK-STAT3 pathway, Data analysis, particularly for cytokine signaling pathways and Revising the manuscript.Jianfei Wen: Execution of rescue experiments and immunofluorescence assays, Data analysis and interpretation and Revising the manuscript.Heda Zhang: Design and execution of experimental work, Data analysis and interpretation, particularly for cytokine signaling and Revising the manuscript.Meiping Shen and Yan Si: As corresponding authors, they ensure the entire team approves the manuscript and the integrity of data, figures, and materials, ensuring reproducibility and transparency in all aspects of the research.All authors reviewed the manuscript. 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SEMIN CANCER BIOL 2017, 45 :13-22. Jin W: Role of JAK/STAT3 Signaling in the Regulation of Metastasis, the Transition of Cancer Stem Cells, and Chemoresistance of Cancer by Epithelial-Mesenchymal Transition . CELLS-BASEL 2020, 9 (1). Guo H, Xiao Y, Yuan Z, Yang X, Chen J, Chen C, Wang M, Xie L, Chen Q, Tong Y et al : Inhibition of STAT3(Y705) phosphorylation by Stattic suppresses proliferation and induces mitochondrial-dependent apoptosis in pancreatic cancer cells . CELL DEATH DISCOV 2022, 8 (1):116. Wang F, Wang X, Li J, Lv P, Han M, Li L, Chen Z, Dong L, Wang N, Gu Y: CircNOL10 suppresses breast cancer progression by sponging miR-767-5p to regulate SOCS2/JAK/STAT signaling . J BIOMED SCI 2021, 28 (1):4. Pandey R, Bakay M, Hakonarson H: SOCS-JAK-STAT inhibitors and SOCS mimetics as treatment options for autoimmune uveitis, psoriasis, lupus, and autoimmune encephalitis . FRONT IMMUNOL 2023, 14 :1271102. Morris R, Kershaw NJ, Babon JJ: The molecular details of cytokine signaling via the JAK/STAT pathway . PROTEIN SCI 2018, 27 (12):1984-2009. Cheng C, Wang P, Yang Y, Du X, Xia H, Liu J, Lu L, Wu H, Liu Q: Smoking-Induced M2-TAMs, via circEML4 in EVs, Promote the Progression of NSCLC through ALKBH5-Regulated m6A Modification of SOCS2 in NSCLC Cells . ADV SCI 2023, 10 (22):e2300953. Chen Q, Zheng W, Guan J, Liu H, Dan Y, Zhu L, Song Y, Zhou Y, Zhao X, Zhang Y et al : SOCS2-enhanced ubiquitination of SLC7A11 promotes ferroptosis and radiosensitization in hepatocellular carcinoma . CELL DEATH DIFFER 2023, 30 (1):137-151. Chen M, Wei L, Law CT, Tsang FH, Shen J, Cheng CL, Tsang LH, Ho DW, Chiu DK, Lee JM et al : RNA N6-methyladenosine methyltransferase-like 3 promotes liver cancer progression through YTHDF2-dependent posttranscriptional silencing of SOCS2 . HEPATOLOGY 2018, 67 (6):2254-2270. Letellier E, Schmitz M, Baig K, Beaume N, Schwartz C, Frasquilho S, Antunes L, Marcon N, Nazarov PV, Vallar L et al : Identification of SOCS2 and SOCS6 as biomarkers in human colorectal cancer . BRIT J CANCER 2014, 111 (4):726-735. Zhang Q, Wei T, Yan L, Zhu S, Jin W, Bai Y, Zeng Y, Zhang X, Yin Z, Yang J et al : Hypoxia-Responsive lncRNA AC115619 Encodes a Micropeptide That Suppresses m6A Modifications and Hepatocellular Carcinoma Progression . CANCER RES 2023, 83 (15):2496-2512. Additional Declarations No competing interests reported. Supplementary Files FigureS1.tif FigureS2.tif Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-5649270","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":391463355,"identity":"f4b93920-549c-4b62-a124-3bdb4d574c78","order_by":0,"name":"Lijun Zhang","email":"","orcid":"","institution":"The First Affiliated Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lijun","middleName":"","lastName":"Zhang","suffix":""},{"id":391463359,"identity":"9e773ce3-6164-407a-9fda-291a7cdea465","order_by":1,"name":"Chengyuan Li","email":"","orcid":"","institution":"The First Affiliated Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chengyuan","middleName":"","lastName":"Li","suffix":""},{"id":391463365,"identity":"dcabe704-8855-4efc-8dd2-6d4799c08fcc","order_by":2,"name":"Jianing Zhou","email":"","orcid":"","institution":"The First Affiliated Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jianing","middleName":"","lastName":"Zhou","suffix":""},{"id":391463367,"identity":"bf48059a-8fc3-4551-adc4-38ed1ce4af2e","order_by":3,"name":"Lin Li","email":"","orcid":"","institution":"Jiangsu Province Hospital","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Li","suffix":""},{"id":391463369,"identity":"dba01aea-8975-48be-960d-a126c2c3828d","order_by":4,"name":"Xiang Zhang","email":"","orcid":"","institution":"The First Affiliated Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Zhang","suffix":""},{"id":391463373,"identity":"610b3445-a591-4b8a-ba4b-5d6ae9f623e3","order_by":5,"name":"Haisheng Fang","email":"","orcid":"","institution":"The First Affiliated Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Haisheng","middleName":"","lastName":"Fang","suffix":""},{"id":391463375,"identity":"6f4ecaff-9456-4955-bad3-b0b406102682","order_by":6,"name":"Jingsheng Cai","email":"","orcid":"","institution":"The First Affiliated Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jingsheng","middleName":"","lastName":"Cai","suffix":""},{"id":391463378,"identity":"51c10059-72af-4a3b-8ee5-6f34fa806ece","order_by":7,"name":"Houchao Tong","email":"","orcid":"","institution":"The First Affiliated Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Houchao","middleName":"","lastName":"Tong","suffix":""},{"id":391463379,"identity":"b5c714dd-6778-45a8-8b39-5aea9b4471e1","order_by":8,"name":"Jianfei Wen","email":"","orcid":"","institution":"The First Affiliated Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jianfei","middleName":"","lastName":"Wen","suffix":""},{"id":391463380,"identity":"ce054c90-f243-4b1d-a9c1-050fa6e776a2","order_by":9,"name":"Heda Zhang","email":"","orcid":"","institution":"The First Affiliated Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Heda","middleName":"","lastName":"Zhang","suffix":""},{"id":391463381,"identity":"c651ce61-4ea4-4fb0-8cb9-eb1ec4d92f4d","order_by":10,"name":"Meiping Shen","email":"","orcid":"","institution":"The First Affiliated Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Meiping","middleName":"","lastName":"Shen","suffix":""},{"id":391463382,"identity":"3ff33659-9c5e-4ab3-93ac-3d2db05f1222","order_by":11,"name":"Yan Si","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYBACA3Yg8cDAAkgyNgAJGx5+/gYCWpiBRIKBBExLmozkjAPEaGGQgPEP2xg0JODXYs7MY/ggoUAisYH/cJvEzx3neQwYDjB++JiDW4tlM4+xAdBhiQ0SiW2SvWdu85gzNzBLztyGx2GHecwkIFoY2yR4227zWDYcYGPmxa/F/AdYC//BNsm/bed4DA4kENRixgDWwpDYJs3bdoCwFstmtmKQw4yBfmm2lm1L5pGccbAZr1/M2Zs3fvjwx0a2gf/4w5tv2+zs+fmbD374iEcLDDjuP8DAAo0dcJwSBvZAzPyBKKWjYBSMglEw4gAAyu9NHjkxj0gAAAAASUVORK5CYII=","orcid":"","institution":"The First Affiliated Hospital of Nanjing Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yan","middleName":"","lastName":"Si","suffix":""}],"badges":[],"createdAt":"2024-12-15 21:23:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5649270/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5649270/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71870139,"identity":"69d7f565-9047-44d6-a84a-dcb52ddc931e","added_by":"auto","created_at":"2024-12-19 10:08:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1882795,"visible":true,"origin":"","legend":"\u003cp\u003eUBE2T expression and its clinical significance in PTC. \u003cstrong\u003eA\u003c/strong\u003eAnalysis of UBE2T mRNA expression across 33 tumor types using data from TCGA, TARGET, and GTEx databases. \u003cstrong\u003eB\u003c/strong\u003e Differential analysis of UBE2T expression in 59 paired PTC and adjacent normal tissues from the TCGA database. \u003cstrong\u003eC\u003c/strong\u003eqRT-PCR results showing significantly higher UBE2T mRNA expression in 100 pairs of PTC tissues compared to adjacent non-tumor tissues. \u003cstrong\u003eD\u003c/strong\u003e Western blot analysis of UBE2T protein expression in 20 paired PTC and adjacent non-tumor tissues. \u003cstrong\u003eE\u003c/strong\u003e IHC staining and scoring showing increased UBE2T expression in tumor tissues versus adjacent non-tumor tissues. \u003cstrong\u003eF\u003c/strong\u003e Kaplan-Meier survival analysis indicating that low UBE2T expression is associated with longer DFI. \u003cstrong\u003eG\u003c/strong\u003e Correlation of UBE2T expression with advanced tumor stage, lymph node metastasis, and extraglandular invasion.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5649270/v1/1df80fcf9e3677af7123f1dd.png"},{"id":71870109,"identity":"39190069-613f-42bf-af6c-9cbfd9762693","added_by":"auto","created_at":"2024-12-19 10:08:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2552409,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of UBE2T overexpression on PTC cell proliferation and migration. \u003cstrong\u003eA\u003c/strong\u003e Western blot analysis confirmed a significant increase in UBE2T protein expression in TPC-1 and KTC-1 cells following overexpression.\u003cstrong\u003eB\u003c/strong\u003e RT-qPCR results demonstrated successful overexpression of UBE2T mRNA in TPC-1 and KTC-1 cells. \u003cstrong\u003eC\u003c/strong\u003e Colony formation assays showed that UBE2T overexpression significantly increased the number of colonies formed by TPC-1 and KTC-1 cells, indicating enhanced proliferative capacity.\u003cstrong\u003e D\u003c/strong\u003e CCK-8 assays revealed that UBE2T overexpression significantly promoted cell proliferation in TPC-1 and KTC-1 cells over time. \u003cstrong\u003eE\u003c/strong\u003e Wound healing assays demonstrated that UBE2T overexpression enhanced the migratory ability of TPC-1 and KTC-1 cells, as indicated by faster wound closure rates. \u003cstrong\u003eF\u003c/strong\u003e Transwell migration assays showed that UBE2T overexpression significantly promoted both migration and invasion of TPC-1 and KTC-1 cells, evidenced by more cells traversing the membrane.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5649270/v1/f155b9cd5d6cc93916659e01.png"},{"id":71870665,"identity":"f17a3927-dd65-4997-8a5b-3517be2ba4d4","added_by":"auto","created_at":"2024-12-19 10:16:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2482075,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of UBE2T knockdown on PTC cell proliferation and migration. \u003cstrong\u003eA\u003c/strong\u003e Western blot analysis confirmed a significant reduction in UBE2T protein expression in TPC-1 and KTC-1 cells following knockdown. \u003cstrong\u003eB\u003c/strong\u003eRT-qPCR results demonstrated effective silencing of UBE2T mRNA expression in TPC-1 and KTC-1 cells. \u003cstrong\u003eC\u003c/strong\u003e Colony formation assays showed that UBE2T knockdown significantly reduced the number of colonies formed by TPC-1 and KTC-1 cells, indicating suppressed proliferative capacity. \u003cstrong\u003eD\u003c/strong\u003e CCK-8 assays revealed that UBE2T knockdown significantly decreased cell proliferation in TPC-1 and KTC-1 cells over time. \u003cstrong\u003eE\u003c/strong\u003e Wound healing assays demonstrated that UBE2T knockdown markedly reduced the migratory ability of TPC-1 and KTC-1 cells, as indicated by slower wound closure rates. \u003cstrong\u003eF\u003c/strong\u003e Transwell migration assays showed that silencing UBE2T significantly suppressed both migration and invasion of TPC-1 and KTC-1 cells, evidenced by fewer cells traversing the membrane.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5649270/v1/f1a5624ea5cfdf39839e7eb3.png"},{"id":71870666,"identity":"5a483fcf-5784-416b-abe2-54c7157ee719","added_by":"auto","created_at":"2024-12-19 10:16:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1347907,"visible":true,"origin":"","legend":"\u003cp\u003eUBE2T promotes PTC progression via the JAK1-STAT3 signaling pathway.\u003cstrong\u003e A\u003c/strong\u003e GSEA showed significant enrichment of the JAK/STAT3 pathway in the high UBE2T expression group. \u003cstrong\u003eB\u003c/strong\u003e Enrichment plot of the JAK/STAT3 pathway in PTC samples with high UBE2T expression. \u003cstrong\u003eC\u003c/strong\u003e UBE2T overexpression increased mRNA levels of JAK/STAT3 target genes (BCL-2, CCND1, VEGFA). \u003cstrong\u003eD\u003c/strong\u003e UBE2T knockdown reduced mRNA levels of BCL-2, CCND1, and VEGFA. \u003cstrong\u003eE\u003c/strong\u003e WB showed elevated phosphorylation of JAK1 and STAT3 in UBE2T-overexpressing cells. \u003cstrong\u003eF\u003c/strong\u003eCorrelation analysis revealed a strong negative relationship between UBE2T and SOCS2 expression (r = -0.42, p \u0026lt; 0.05). \u003cstrong\u003eG\u003c/strong\u003e Co-IP confirmed the interaction between UBE2T and SOCS2.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5649270/v1/c2778ba63edfc4d961ece8ef.png"},{"id":71870111,"identity":"018daf15-b2ed-4636-8398-198dabae9d92","added_by":"auto","created_at":"2024-12-19 10:08:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2986277,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary analyses of UBE2T and JAK/STAT signaling. \u003cstrong\u003eA\u003c/strong\u003eCo-IP showed no direct interaction between UBE2T and JAK1. \u003cstrong\u003eB\u003c/strong\u003e Co-IP showed no direct interaction between UBE2T and STAT3. \u003cstrong\u003eC-F\u003c/strong\u003e Correlation analysis between UBE2T and SOCS4,SOCS5,SOCS6 and SOCS7.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5649270/v1/9e85a8e65bbd324329058a11.png"},{"id":71870670,"identity":"81a3dda7-060c-4946-887e-ed5843864501","added_by":"auto","created_at":"2024-12-19 10:16:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3371606,"visible":true,"origin":"","legend":"\u003cp\u003eSOCS2 mediates the effects of UBE2T on PTC progression. \u003cstrong\u003eA\u003c/strong\u003eUBE2T overexpression promoted cell proliferation, partially reversed by SOCS2 upregulation. \u003cstrong\u003eB\u003c/strong\u003e UBE2T overexpression enhanced invasion, mitigated by SOCS2 upregulation. \u003cstrong\u003eC\u003c/strong\u003e Wound healing assays showed SOCS2 upregulation reduced UBE2T-induced migration. \u003cstrong\u003eD\u003c/strong\u003e UBE2T-induced upregulation of JAK1/STAT3 target genes was partially reversed by SOCS2 upregulation.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5649270/v1/0da82570f99d7b22bfeca7f1.png"},{"id":74834956,"identity":"c19c7274-cf6e-4cd1-a5bd-3c3f0d0293e8","added_by":"auto","created_at":"2025-01-27 11:32:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19264103,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5649270/v1/33016980-e254-4b1b-af10-995c76f207a1.pdf"},{"id":71872009,"identity":"d5fb1b91-1b5d-4c7a-a71e-c3b0b096a619","added_by":"auto","created_at":"2024-12-19 10:24:54","extension":"tif","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":9757928,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-5649270/v1/aed88c345f6a2064496fdb0c.tif"},{"id":71870671,"identity":"5df36ca0-8178-4dce-906f-84e1afd9207b","added_by":"auto","created_at":"2024-12-19 10:16:58","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18530292,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-5649270/v1/52f28d5ced14d719fbebd34b.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"UBE2T promotes PTC progression by activating the JAK/STAT3 pathway via negative regulation of SOCS2","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThyroid cancer is the most prevalent endocrine malignancy, with a dramatic rise in incidence over the past two decades, making it the eighth most common malignancy globally and the fourth most frequent cancer among women[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This surge is primarily attributed to the increasing prevalence of PTC, which accounts for the majority of thyroid cancer cases. While most PTCs exhibit indolent growth and favorable prognosis, a subset (10\u0026ndash;15%) demonstrates aggressive phenotypes characterized by extrathyroidal invasion and cervical lymph node metastasis, which are strongly associated with poor clinical outcomes. Despite extensive efforts to elucidate the molecular underpinnings of PTC aggressiveness, particularly through studies on pathways involving BRAF, TERT, and MAPK[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], effective methods for predicting or mitigating its invasive behavior remain elusive.\u003c/p\u003e \u003cp\u003eUBE2T, a member of the E2 enzyme family, has emerged as a critical oncogenic player in multiple malignancies, such as cholangiocarcinoma[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], colorectal[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], esophageal[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], lung[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and pancreatic cancers[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. By promoting proliferation, migration, and invasion through diverse molecular pathways, UBE2T has been highlighted as a potential therapeutic target. Our preliminary findings revealed that UBE2T is significantly upregulated in PTC tissues compared to normal thyroid tissues, correlating with aggressive features such as lymph node metastasis and extrathyroidal invasion. However, the molecular mechanisms underlying UBE2T-driven PTC progression remain poorly defined.\u003c/p\u003e \u003cp\u003eThe development of tumors is often driven by signaling dysregulation resulting from gene mutations or abnormal gene expression. These aberrantly activated signaling pathways are core mechanisms that drive cell proliferation, immune evasion, anti-apoptosis, and invasion/metastasis[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. For example, pathways such as MAPK, PI3K/Akt, and JAK/STAT, when aberrantly activated, can continuously transmit \"growth\" signals, enabling tumor cells to escape normal regulation.\u003c/p\u003e \u003cp\u003eIn PTC, the most commonly dysregulated pathway is the MAPK signaling pathway[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Approximately 70%-80% of PTC cases exhibit BRAF\u003csup\u003eV600E\u003c/sup\u003e mutations and RAS gene mutations, leading to sustained activation of MEK and ERK, which promotes tumor progression. It remains an open question whether UBE2T also promotes tumor progression in PTC through modulation of these classical signaling pathways. This is a key issue that warrants further investigation.\u003c/p\u003e \u003cp\u003eIn this study, we aimed to elucidate the molecular mechanisms by which UBE2T mediates PTC aggressiveness. Using TCGA and GEO databases, along with clinical validation, we analyzed UBE2T expression and its association with clinicopathological features in PTC. Functional assays were conducted to assess the effects of UBE2T overexpression and knockdown on PTC cell behaviors. Mechanistically, we demonstrated that UBE2T promotes PTC progression by activating the JAK/STAT3 signaling pathway through the suppression of SOCS2, a key inhibitory molecule in the pathway. These findings were further validated through co-IP, IF, and rescue experiments, highlighting UBE2T as a potential therapeutic target in aggressive PTC.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePublic data\u003c/h2\u003e \u003cp\u003eWe downloaded the uniformly standardized pan-cancer dataset from the UCSC (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://xenabrowser.net/\u003c/span\u003e\u003cspan address=\"https://xenabrowser.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) database, which includes TCGA, TARGET, and GTEx. We filtered out samples with an expression level of 0 and excluded cancer types with fewer than three samples within a single cancer group. Ultimately, expression data from 34 cancer types were obtained for single-gene pan-cancer analysis.\u003c/p\u003e \u003cp\u003eAdditionally, we retrieved the mRNA expression matrix file for TCGA-THCA from the Genomic Data Sharing (GDS) portal (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the mRNA expression matrix file for normal thyroid tissue from GTEx for subsequent correlation analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eClinical Samples\u003c/h3\u003e\n\u003cp\u003eThis study utilized 100 pairs of tissue samples collected from patients who underwent surgery at Jiangsu Provincial People's Hospital (The First Affiliated Hospital of Nanjing Medical University) between January 2022 and December 2023. The study was approved by the hospital's ethics committee (Ethics Approval No. 2023-SR-425), and all participants provided written informed consent.\u003c/p\u003e\n\u003ch3\u003eQuantitative RT–PCR\u003c/h3\u003e\n\u003cp\u003eAdd 0.5-1 mL of Trizol reagent to an appropriate amount of tissue or culture dish, followed by the addition of 1/5 the volume of chloroform. Incubate for 10 minutes and centrifuge at 12,000g for 15 minutes at 4\u0026deg;C. Transfer the upper aqueous phase to a new tube, add an equal volume of isopropanol, shake, and incubate for 10 minutes. Centrifuge under the same conditions, discard the supernatant, and retain the pellet. Add 1 mL of 75% ethanol, centrifuge at 12,000g for 10 minutes, and repeat the wash once. Retain the pellet to obtain total RNA. The RNA concentration was measured using a Nanodrop (Thermo Fisher). RNA was reverse transcribed into cDNA using the FastKing gDNA Dispelling RT SuperMix (TIANGEN, KR118-01) kit, with 500 ng of RNA used in each reverse transcription reaction. qPCR was performed using SuperReal PreMix Plus (SYBR Green) (TIANGEN, FP205), and data were collected and analyzed using the Applied Biosystems PCR System (StepOnePlus Real-Time PCR System, Thermo Fisher).\u003c/p\u003e\n\u003ch3\u003eWestern Blot\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern Blot\u003c/div\u003e \u003cp\u003eProtein extraction from cells or tissue samples was performed under low-temperature conditions. An appropriate amount of Protease Inhibitor Cocktail (100X) (CST, 5871) was added to RIPA lysis buffer (Servicebio, G2002). The samples were sonicated at 30% power for 20 seconds, followed by a 20-second ice bath, repeated three times. The samples were then centrifuged at 15,000 rpm for 20 minutes at 4\u0026deg;C. The supernatant was collected, and protein concentration was determined using a BCA assay kit. Protein loading buffer (4:1 volume ratio) was added to the protein extract in a pre-chilled EP tube and thoroughly mixed. The samples were denatured by heating in a 100\u0026deg;C water bath for 10 minutes. Protein separation was performed using 7.5% (Epizyme Biotech, PG211) or 10% SDS-PAGE gel (Epizyme Biotech, PG212), and the proteins were transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk solution for 1.5 hours, followed by incubation with the corresponding primary antibody at 4\u0026deg;C overnight. On the following day, the membrane was washed three times with TBST, each wash for 10 minutes. The membrane was then incubated with the secondary antibody at room temperature for 2 hours, washed again with TBST three times, each wash for 10 minutes, and finally exposed.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry (IHC)\u003c/h3\u003e\n\u003cp\u003eParaffin-embedded tissue sections (4 \u0026micro;m) were baked at 65\u0026deg;C for 1 hour, deparaffinized in xylene, and rehydrated in a gradient of ethanol. Antigen retrieval was performed by microwaving the sections in pH 6.0 sodium citrate buffer for 10 minutes at high temperature, followed by cooling and washing the sections three times with PBS. The sections were incubated with 3% hydrogen peroxide solution at room temperature for 10 minutes to block endogenous peroxidase activity, and then blocked with 5% normal goat serum at room temperature for 30 minutes.\u003c/p\u003e \u003cp\u003eThe UBE2T antibody (Thermo Fisher, PA5-28464) was applied and incubated overnight at 4\u0026deg;C. The next day, after PBS washing, biotinylated secondary antibody was added and incubated at room temperature for 30 minutes. After another PBS wash, HRP-conjugated streptavidin was added and incubated for 30 minutes. DAB was used for color development for 2\u0026ndash;5 minutes, and the reaction was terminated by rinsing with tap water. The sections were counterstained with hematoxylin for 1 minute, dehydrated through a gradient of ethanol, cleared in xylene, and mounted. The results were observed under a microscope.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLentiviral Infection\u003c/h2\u003e \u003cp\u003eThe TPC-1 cells (FuHeng BioLogy, FH1039) and KTC-1 cells (Servicebio, STCC12507P) were adjusted to a concentration of 6\u0026times;104 cells/mL and seeded at 2 mL per well in a 6-well plate. The infection was performed when the cell density reached 80%-90% the following day. Before infection, lentivirus suspension (purchased from Genechem) was prepared, and the required viral amount was calculated based on the MOI. The viral suspension was mixed with complete medium, gently mixed, and added to the cell culture plate.The cells were incubated at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for 8\u0026ndash;12 hours. After the incubation, the fresh complete medium was replaced, and the cells continued to be cultured. After 48\u0026ndash;72 hours of infection, puromycin was used to select positive cell clones.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePlasmid Transfection\u003c/h3\u003e\n\u003cp\u003eBefore the experiment, PTC cells were seeded in 6-well plates at 60%-80% confluence to ensure they were in a good growth condition. 0.5-2 \u0026micro;g of plasmid DNA (purchased from Genechem) was added per well. The plasmid DNA was diluted in serum-free medium and mixed with the transfection reagent Lipo3000 (Biosharp, BL632A). The plasmid DNA and transfection reagent were gently mixed and allowed to stand at room temperature for 15\u0026ndash;20 minutes to form a DNA-transfection reagent complex.The original culture medium was removed, and serum-free medium was added. The DNA-transfection reagent complex was then added to the cells, gently mixed to ensure uniform distribution, and the cells were incubated in a 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for 4\u0026ndash;6 hours. Afterward, the medium was replaced with complete medium containing serum, and the cells were further cultured.\u003c/p\u003e\n\u003ch3\u003eColony Formation Assay\u003c/h3\u003e\n\u003cp\u003eCells were seeded at a density of 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well in a 6-well plate. After the cells adhered, they were transfected with plasmids. After 48 hours, cells in the logarithmic growth phase were collected, counted using a cell counting chamber, and approximately 400 cells were seeded per well. The cells were then cultured in a 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for 14 days, with medium changes as needed. After 14 days, cells were fixed with 3% paraformaldehyde and stained with crystal violet. The colonies were photographed and counted.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCCK-8 Assay\u003c/h2\u003e \u003cp\u003eCells were seeded at 2\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells per well in 100 \u0026micro;L medium in a 96-well plate. After 24 hours of incubation at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e, 10 \u0026micro;L of CCK-8 solution (ApexBio, K1018) was added to each well. The cells were incubated in the incubator for 1.5 hours, and the absorbance at 450 nm was measured.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWound Healing Assay\u003c/h2\u003e \u003cp\u003eThree horizontal lines were drawn evenly on the bottom of a 6-well plate. After seeding 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well and allowing them to adhere, cells were transfected with plasmids. After 48 hours, a 20 \u0026micro;L pipette tip was used to create two vertical lines in each well. PBS was used to wash away floating cells, and wound healing was observed for 12\u0026ndash;48 hours. A camera was used to capture images of the wound healing process.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTranswell Assay\u003c/h2\u003e \u003cp\u003eCells were seeded at a density of 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well in a 6-well plate. After allowing the cells to adhere, they were transfected with plasmids. After 48 hours, cells in the logarithmic growth phase were collected, counted using a cell counting chamber, and resuspended in serum-free medium. The cell concentration was adjusted to 10^5 cells/mL and mixed thoroughly.For the invasion assay, Matrigel was added to the upper chamber of the Transwell. In each well, 500 \u0026micro;L of complete medium was added to the lower chamber, and 200 \u0026micro;L of the cell suspension was added to the upper chamber. The plate was incubated at 37\u0026deg;C in a cell culture incubator, with TPC-1 cells incubated for 24 hours and KTC-1 cells for 18 hours. After incubation, the chamber was removed, washed twice with PBS, fixed with 4% paraformaldehyde for 15 minutes, and stained with 0.1% crystal violet for 20 minutes. Unbound crystal violet and non-migrated cells on the upper side of the chamber were removed. Finally, the cells were observed and counted under a microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCo-IP Assay\u003c/h2\u003e \u003cp\u003eUBE2T-overexpressing or UBE2T-knockdown cells were lysed in IP lysis buffer (Thermo Fisher, 87787) and incubated on ice for 30 minutes. After centrifugation at 12,000 rpm for 15 minutes at 4\u0026deg;C, the cell lysate was incubated with primary antibodies against SOCS2 (Abcam, ab109245) and Flag (Beyotime, AF519) at 4\u0026deg;C overnight. The immunocomplex was then incubated with Dynabeads magnetic beads (Thermo Fisher, 10003D) for 4 hours at 4\u0026deg;C. After washing the immunocomplex three times, protein sample buffer was added, and the samples were boiled for denaturation before performing WB analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAnimal Experiments\u003c/h2\u003e \u003cp\u003e4-6-week-old BALB/c nude mice (Charles River, CAnN.Cg-\u003cem\u003eFoxn1\u003c/em\u003e\u003csup\u003e\u003cem\u003enu\u003c/em\u003e\u003c/sup\u003e/Crl) were housed in an SPF environment. After culturing cells to the logarithmic growth phase (80\u0026ndash;90% confluence), the cells were digested with trypsin to prepare a cell suspension (4\u0026times;10\u003csup\u003e7\u003c/sup\u003e cells/mL), which was kept on ice. For subcutaneous implantation, 100\u0026micro;L of cell suspension was injected subcutaneously into the axillary region of nude mice, and tumor growth was monitored regularly. When tumor volume reached approximately 2000 mm\u0026sup3;, samples were collected. For lung metastasis experiments, the cell suspension (1\u0026ndash;2\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/mL) was injected via the tail vein (100\u0026ndash;200\u0026micro;L per mouse). After injection, the mice were kept in a controlled temperature and humidity environment, and tumor metastasis was monitored using fluorescence imaging over a 2\u0026ndash;6 week period. All animal experiments were conducted in accordance with the guidelines of the Animal Ethics Committee of Nanjing Medical University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eIF Staining:\u003c/h2\u003e \u003cp\u003eParaffin-embedded tissue sections (4\u0026ndash;6\u0026micro;m) were deparaffinized and rehydrated, followed by antigen retrieval with citrate buffer (pH 6.0) using high-pressure cooking. After blocking with 5% normal serum for 30 minutes, the sections were incubated with primary antibodies against UBE2T (GeneTex, GTX83452) and SOCS2 (Abcam, ab109245) in a humidity chamber at 4\u0026deg;C overnight. The next day, the sections were washed with PBS and incubated with corresponding fluorescent-labeled secondary antibodies at room temperature for 1 hour, protected from light. Nuclei were stained with DAPI, and after PBS washing, the sections were mounted with anti-fluorescence quenching mounting medium. Images were captured using a laser confocal microscope. The entire process was carried out under strict light protection to prevent fluorescence quenching.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eExpression differences between normal and tumor samples in each tumor were calculated using R software (version 3.6.4). Statistical significance was analyzed using the unpaired Wilcoxon Rank Sum and Signed Rank Tests. Student's t-test was used for statistical comparisons between two experimental conditions with unpaired samples, while the Wilcoxon Rank Sum test was used for all paired t-tests. One-way analysis of variance (ANOVA) was used for comparisons among multiple experimental groups. Disease-Free Interval (DFI) analysis was performed using Kaplan-Meier plots. *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 indicate statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003ch3\u003eUBE2T is Overexpressed in PTC and Correlates with Aggressive Characteristics\u003c/h3\u003e\n\u003cp\u003eTo explore the potential role of UBE2T in the development of PTC, we conducted a comprehensive analysis of UBE2T expression in tumor and corresponding normal tissues across various cancers using gene expression data from the TCGA, TARGET, and GTEx databases. The results indicated that UBE2T mRNA expression was significantly upregulated in 33 different tumor types (Figure 1A). Further differential analysis of RNA-seq data from 59 paired PTC samples in the TCGA database confirmed that UBE2T expression was significantly elevated in PTC tissues compared to normal tissues (Figure 1B). Subsequently, we performed qRT-PCR to assess UBE2T expression in 100 pairs of PTC and adjacent non-tumor tissues that met inclusion and exclusion criteria at our institution. The results showed that UBE2T expression was significantly higher in PTC tissues than in adjacent non-tumor tissues, with a statistically significant difference (Figure 1C). Western blotting analysis of UBE2T protein expression in 20 paired PTC and adjacent non-tumor tissues also revealed significantly higher protein levels in PTC tissues (Figure 1D).\u003c/p\u003e\n\u003cp\u003eTo further investigate the potential clinical and pathological effects of UBE2T, we examined UBE2T expression in human PTC samples by IHC. The IHC scores confirmed that UBE2T was more highly expressed in tumor tissues compared to adjacent non-tumor tissues (Figure 1E). Kaplan-Meier survival analysis showed that patients with low UBE2T expression had significantly longer DFI compared to those with high UBE2T expression (Figure 1F). Notably, higher UBE2T expression was closely associated with advanced tumor staging, lymph node metastasis, and extraglandular invasion (Figure 1G).\u003c/p\u003e\n\u003ch3\u003eUBE2T Promotes PTC Cell Proliferation and Migration\u003c/h3\u003e\n\u003cp\u003eTo further investigate the regulatory role of UBE2T in PTC, we constructed stable UBE2T-overexpressing PTC cell lines (TPC-1 and KTC-1) using lentivirus. The transfection efficiency was evaluated by Western blot and RT-qPCR (Figure 2A, B). We also used plasmids to effectively knock down UBE2T expression (Figure 3A, B). As expected, in vitro experiments showed that overexpression of UBE2T promoted PTC cell proliferation compared to control cells (Figure 2C, D). In contrast, silencing UBE2T inhibited proliferation in TPC-1 and KTC-1 cells (Figure 3C, D). To assess the pro-migratory effect of UBE2T, we conducted wound healing and Transwell migration assays. Overexpression of UBE2T significantly enhanced the invasion and migration of TPC-1 and KTC-1 cells (Figure 2E, F), while UBE2T knockdown inhibited their invasion and migration (Figure 3E, F). Overall, these in vitro results suggest that UBE2T is a positive regulator of PTC cell proliferation and migration.\u003c/p\u003e\n\u003ch3\u003eUBE2T Activates the JAK1/STAT3 Signaling Pathway in PTC\u003c/h3\u003e\n\u003cp\u003eTo further explore the mechanism by which UBE2T promotes thyroid cancer cell proliferation, invasion, and migration, we divided the samples into high (\u0026ge;50% expression) and low (\u0026lt;50% expression) UBE2T expression groups and performed single-gene GSEA analysis. The results suggested that the JAK/STAT3 signaling pathway could be a key pathway through which UBE2T promotes PTC cell proliferation, invasion, and migration (Figure 4A, B).\u003c/p\u003e\n\u003cp\u003eAfter overexpressing UBE2T in TPC-1 and KTC-1 cells, we measured the mRNA expression of JAK/STAT3 pathway target genes (BCL-2, CCND1, VEGFA) by qRT-PCR. The results showed that UBE2T overexpression upregulated the expression of these downstream target genes compared to controls (Figure 4C), while UBE2T knockdown significantly reduced the expression of BCL-2, CCND1, and VEGFA (Figure 4D). Western blotting of phosphorylated JAK1, JAK2, and STAT3 proteins showed significantly increased phosphorylation of JAK1 and STAT3 in UBE2T-overexpressing cells compared to controls (Figure 4E), suggesting that UBE2T may promote carcinogenesis through activation of the JAK1-STAT3 signaling pathway.\u003c/p\u003e\n\u003cp\u003eTo determine whether UBE2T activates this pathway by directly interacting with JAK1 or STAT3, we conducted Co-IP experiments. Unfortunately, the results did not support an interaction between UBE2T and these proteins (Figure S1A-B).\u003c/p\u003e\n\u003cp\u003eTo identify potential interacting molecules of UBE2T, we performed a correlation analysis using PTC samples from the TCGA-THCA dataset. The results indicated a significant negative correlation between UBE2T and SOCS2 expression (r = -0.42, p \u0026lt; 0.05) within the SOCS family (Figure 4F and Figure S1C-F). SOCS family members are important negative regulators of cytokine signaling, playing a role in feedback inhibition of the JAK-STAT pathway. Co-IP experiments confirmed that SOCS2 was successfully detected in UBE2T immunocomplexes, whereas no related signal was observed in the negative control (IgG group) (Figure 4G). This suggests that UBE2T interacts with SOCS2 to participate in relevant signal regulatory mechanisms.\u003c/p\u003e\n\u003ch3\u003eUBE2T Activates JAK1/STAT3 Pathway by Downregulating SOCS2 to Promote PTC Progression\u003c/h3\u003e\n\u003cp\u003eTo further investigate whether UBE2T promotes PTC cell proliferation and invasion through its interaction with SOCS2, we performed rescue experiments. The results showed that overexpression of UBE2T promoted the proliferation, invasion, and migration of PTC cell lines, and upregulation of SOCS2 partially reversed these effects (Figure 5A-C). In contrast, knockdown of UBE2T inhibited the proliferation, invasion, and migration of TPC-1 and KTC-1 cells, and suppression of SOCS2 expression partially reversed these inhibitory effects (Figure S2A-C). Moreover, we found that overexpression of UBE2T upregulated the expression of downstream target genes in the JAK1/STAT3 pathway, and upregulation of SOCS2 partially reversed this effect (Figure 5D). Conversely, UBE2T knockdown reduced the expression of these target genes, and suppression of SOCS2 partially reversed this reduction (Figure S2D).\u003c/p\u003e\n\u003cp\u003eTo further confirm the pro-cancer role of UBE2T in PTC, we conducted in vivo experiments. The results showed that tumors from nude mice with upregulated UBE2T expression had significantly larger volumes and weights compared to controls (Figure 6A). In contrast, tumors from nude mice with UBE2T knockdown were significantly smaller than controls (Figure 6B).\u003c/p\u003e\n\u003cp\u003eNext, we used a tail vein injection model to investigate the role of UBE2T in PTC metastasis in vivo. Fluorescence signal intensity measurements revealed that the lungs of mice overexpressing UBE2T exhibited significantly higher average fluorescence signal intensity compared to controls, with a statistically significant difference (Figure 6C). In contrast, mice with UBE2T knockdown showed lower fluorescence signal intensity in their lung tissues compared to controls (Figure 6D). These results suggest that UBE2T promotes PTC tumor proliferation and metastasis in vivo.\u003c/p\u003e\n\u003cp\u003eTo validate the relationship between UBE2T and SOCS2, we performed immunofluorescence staining on PTC tissues. The immunofluorescence results showed that UBE2T and SOCS2 predominantly co-localized in the cytoplasm (Figure 6E). This further supports that in PTC, UBE2T may influence tumor progression through its interaction with SOCS2.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur findings provide novel mechanistic insights into the oncogenic role of UBE2T in the progression of PTC. Through analysis of public datasets and institutional clinical samples, we demonstrated that UBE2T is significantly overexpressed in PTC tissues. Functional assays conducted both in vitro and in vivo revealed that UBE2T promotes malignant phenotypes, including enhanced cellular proliferation and invasion. Mechanistic investigations, incorporating GSEA and Western blot analyses, identified the JAK/STAT3 signaling pathway as a critical downstream effector mediating the tumor-promoting effects of UBE2T.\u003c/p\u003e \u003cp\u003eThe JAK/STAT3 pathway, an essential cellular signal transduction pathway, is widely involved in various cellular processes such as growth, differentiation, proliferation, apoptosis, and immune regulation[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In oncology research, the activation of the JAK/STAT3 pathway is frequently used as a prognostic marker. For example, overexpression of STAT3 is associated with poor prognosis in several solid tumors[\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Additionally, STAT3 can enhance the immunosuppressive functions of tumor-associated macrophages (TAMs), inhibit CD8\u0026thinsp;+\u0026thinsp;T cell activity, and promote the proliferation of regulatory T cells (Tregs), thereby aiding tumor cells in evading immune surveillance[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven the pivotal role of the JAK/STAT3 pathway in tumor development, STAT3 has been considered an important therapeutic target, and several classic STAT3 inhibitors, such as Stattic, have been developed[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Moreover, strategies targeting the inhibition of STAT phosphorylation or blocking its DNA-binding have been explored. The development of STAT inhibitors and JAK kinase inhibitors has become a significant focus in recent cancer drug research.\u003c/p\u003e \u003cp\u003eOur experimental findings show that UBE2T overexpression enhances the phosphorylation of JAK1 and STAT3, thereby activating this pathway. Furthermore, our study highlights the role of the oncogenic factor UBE2T in activating the JAK/STAT3 pathway through SOCS2.\u003c/p\u003e \u003cp\u003ePrevious studies have demonstrated that SOCS2 is a downstream molecule of JAK but exerts regulatory effects on the JAK signaling pathway through a negative feedback mechanism[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. When cytokines such as IL-6, EPO, or GH activate JAK kinases through receptors, JAK phosphorylates specific sites on the receptor, recruiting and activating STAT proteins[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The activated STAT proteins enter the nucleus, bind to the promoter regions of target genes, and induce SOCS2 transcription. Once SOCS2 is produced, it negatively regulates JAK signaling by directly inhibiting JAK kinase activity or promoting the degradation of receptors or JAKs. SOCS2 has a dual role in various tumors, acting both as a tumor suppressor and, in certain contexts, promoting tumor progression[\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In most tumors, including breast cancer[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], colorectal cancer[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and pancreatic cancer[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], SOCS2 exerts a negative regulatory effect, which aligns with its tumor-suppressive role in PTC.\u003c/p\u003e \u003cp\u003eCurrently, there is limited research on the relationship between UBE2T, SOCS2, and JAK/STAT3 in the literature. Nevertheless, our results provide novel evidence emphasizing that UBE2T activates the JAK/STAT3 pathway by negatively regulating SOCS2, thus promoting the progression of PTC. This study has certain limitations, including the need for further clarification of the precise mechanism by which UBE2T regulates SOCS2, as well as the therapeutic potential of STAT3 inhibitors in blocking PTC progression.\u003c/p\u003e \u003cp\u003eIn conclusion, our study demonstrates the critical role of UBE2T in promoting the progression of PTC. UBE2T is highly expressed in PTC and activates the JAK/STAT3 pathway through SOCS2. These findings contribute to a deeper understanding of the molecular mechanisms underlying PTC progression and offer potential diagnostic and therapeutic targets for PTC invasiveness.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eAbbreviation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eFull Name\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003ePTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003epapillary thyroid carcinoma\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eco-IP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eco-immunoprecipitation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eSOCS2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003ecytokine signaling suppressor 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eIF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eimmunofluorescence\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eGDS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003ethe Genomic Data Sharing\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eIHC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eimmunohistochemistry\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eANOVA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eone-way analysis of variance\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eDFI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003edisease-free interval\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eTAMs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003etumor-associated macrophages\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eTregs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eregulatory T cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding Declaration\u003c/h2\u003e\n\u003cp\u003eThis work was supported by Jiangsu Provincial Medical Key Discipline （No. ZDXK202222）.\u003c/p\u003e\n\u003ch2\u003eEthics Approval and Consent to Participate\u003c/h2\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of Jiangsu Province Hospital (Approval No. 2023-SR-425).\u003c/p\u003e\n\u003cp\u003eAll authors have consented to the publication of this manuscript in \u003cem\u003eCancer Cell International\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLijun Zhang: Conception and design of the study；acquisition and analysis of data, Drafting the manuscript and revising it and Ensure data integrity and support for transparency and reproducibility.Chengyuan Li: Acquisition of data from clinical samples and laboratory experiments, analysis and interpretation of results, including JAK-STAT3 pathway activation and Drafting sections of the manuscript and revising it.Jianing Zhou: Acquisition of clinical samples and data, Analysis and interpretation of clinical pathological features and Drafting sections of the manuscript and revising it.Lin Li: Design of experiments, Creation of new software (if applicable) for data analysis and Revising the manuscript and contributing to experimental design.Xiang Zhang: Data collection and analysis, Co-immunoprecipitation experiments and result interpretation and Revising the manuscript.Haisheng Fang: Conception and design of experiments, Interpretation of data related to the JAK-STAT3 signaling pathway and revising the manuscript.Jingsheng Cai: Acquisition of clinical data, Data analysis related to pathological features and Revising the manuscript.Houchao Tong: Conception and design of experiments focused on the JAK-STAT3 pathway, Data analysis, particularly for cytokine signaling pathways and Revising the manuscript.Jianfei Wen: Execution of rescue experiments and immunofluorescence assays, Data analysis and interpretation and Revising the manuscript.Heda Zhang: Design and execution of experimental work, Data analysis and interpretation, particularly for cytokine signaling and Revising the manuscript.Meiping Shen and Yan Si: As corresponding authors, they ensure the entire team approves the manuscript and the integrity of data, figures, and materials, ensuring reproducibility and transparency in all aspects of the research.All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe acknowledge the financial and technical support provided by Jiangsu Provincial Medical Key Discipline ZDXK202222, which made this work possible.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSiegel RL, Miller KD, Wagle NS, Jemal A: \u003cstrong\u003eCancer statistics, 2023\u003c/strong\u003e. \u003cem\u003eCA: A Cancer Journal for Clinicians\u003c/em\u003e 2023, \u003cstrong\u003e73\u003c/strong\u003e(1):17-48.\u003c/li\u003e\n\u003cli\u003eHan B, Zheng R, Zeng H, Wang S, Sun K, Chen R, Li L, Wei W, He J: \u003cstrong\u003eCancer incidence and mortality in China, 2022\u003c/strong\u003e. \u003cem\u003eJournal of the National Cancer Center\u003c/em\u003e 2024.\u003c/li\u003e\n\u003cli\u003eYu P, Qu N, Zhu R, Hu J, Han P, Wu J, Tan L, Gan H, He C, Fang C\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eTERT accelerates BRAF mutant-induced thyroid cancer dedifferentiation and progression by regulating ribosome biogenesis\u003c/strong\u003e. \u003cem\u003eSCI ADV\u003c/em\u003e 2023, 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\u003cstrong\u003e67\u003c/strong\u003e(6):2254-2270.\u003c/li\u003e\n\u003cli\u003eLetellier E, Schmitz M, Baig K, Beaume N, Schwartz C, Frasquilho S, Antunes L, Marcon N, Nazarov PV, Vallar L\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eIdentification of SOCS2 and SOCS6 as biomarkers in human colorectal cancer\u003c/strong\u003e. \u003cem\u003eBRIT J CANCER\u003c/em\u003e 2014, \u003cstrong\u003e111\u003c/strong\u003e(4):726-735.\u003c/li\u003e\n\u003cli\u003eZhang Q, Wei T, Yan L, Zhu S, Jin W, Bai Y, Zeng Y, Zhang X, Yin Z, Yang J\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eHypoxia-Responsive lncRNA AC115619 Encodes a Micropeptide That Suppresses m6A Modifications and Hepatocellular Carcinoma Progression\u003c/strong\u003e. \u003cem\u003eCANCER RES\u003c/em\u003e 2023, \u003cstrong\u003e83\u003c/strong\u003e(15):2496-2512.\u003c/li\u003e\n\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":"UBE2T, thyroid carcinoma, SOCS2, JAK/STAT3","lastPublishedDoi":"10.21203/rs.3.rs-5649270/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5649270/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThyroid cancer, particularly papillary thyroid carcinoma (PTC), is one of the most common malignant tumors of the endocrine system. Malignant biological behaviors such as tumor invasion and cervical lymph node metastasis are closely associated with the prognosis of PTC. To date, no effective method has been identified to accurately predict the invasive biological behavior of PTC.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eThis study aims to investigate the potential molecular mechanisms underlying the high invasiveness of PTC mediated by UBE2T.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe examined the expression of UBE2T in PTC using data from the TCGA and GEO databases and validated these findings in clinical samples from our institution, analyzing clinical pathological features. Subsequently, we explored the impact of UBE2T on the biological behavior of PTC cells through stable overexpression or knockdown of the UBE2T gene. Additionally, we elucidated the potential mechanisms by which UBE2T promotes PTC progression, with a particular focus on its role in activating the JAK-STAT signaling pathway.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOur results demonstrate that UBE2T plays a crucial role in promoting PTC progression by activating the JAK-STAT signaling pathway. Correlation analysis and co-immunoprecipitation (co-IP) experiments identified cytokine signaling suppressor 2 (SOCS2) as a key molecule mediating UBE2T's action in the JAK-STAT pathway. Further rescue experiments and immunofluorescence (IF) assays confirmed that UBE2T promotes PTC progression by negatively regulating SOCS2, thereby activating the JAK-STAT3 pathway.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study reveals the mechanistic role of UBE2T in the high invasiveness of PTC, highlighting its negative regulation of SOCS2 to activate the JAK-STAT3 signaling pathway and drive PTC progression. These findings provide new insights into the mechanisms of PTC invasion and may offer potential therapeutic targets for inhibiting PTC metastasis and recurrence.\u003c/p\u003e","manuscriptTitle":"UBE2T promotes PTC progression by activating the JAK/STAT3 pathway via negative regulation of SOCS2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-19 10:08:37","doi":"10.21203/rs.3.rs-5649270/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":"7e5fe627-1749-4de4-98b0-5ef7343e266a","owner":[],"postedDate":"December 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-06T03:23:12+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-19 10:08:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5649270","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5649270","identity":"rs-5649270","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}