From Data Mining to Functional Validation: UBE2S Regulates Proliferation and Radiosensitivity in Colorectal Cancer via the PI3K/AKT Pathway

From Data Mining to Functional Validation: UBE2S Regulates Proliferation and Radiosensitivity in Colorectal Cancer via the PI3K/AKT Pathway | 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 From Data Mining to Functional Validation: UBE2S Regulates Proliferation and Radiosensitivity in Colorectal Cancer via the PI3K/AKT Pathway Yu Chang, Yu Liu, Yaqin Hao, Jing Guo, Kai Bai, Fei Huang, Yunfeng Hu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7724776/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 Colorectal cancer is the third most common malignant tumor worldwide, accounting for approximately 10% of all newly diagnosed cancer cases annually. UBE2S has been demonstrated to play an oncogenic role in various types of cancer. however, its specific mechanism in colorectal cancer remains poorly understood. Methods In this study, we initially identified the differential expression of UBE2S between colorectal cancer tissues and normal colorectal tissues from patients by analyzing data from TCGA and GEO databases. Meanwhile, UBE2S also exhibits certain significance in pan-cancer. Further studies showed that compared with normal colorectal epithelial cells, UBE2S was highly expressed in colorectal cancer cells and had a certain correlation with immune infiltrating cells. We then investigated the effects of UBE2S knockdown and overexpression on the proliferation and radiotherapy sensitivity of colorectal cancer cells. RNA sequencing analysis revealed that UBE2S was enriched in pathways related to cell cycle, apoptosis, PI3K/AKT signaling, DNA replication, base excision repair, ferroptosis, and non-homologous end joining. Subsequently, we evaluated the changes in PI3K/AKT pathway-related proteins after UBE2S knockout and overexpression, as well as the effect of the PI3K/AKT pathway inhibitor LY294002 on the proliferation of colorectal cancer cells. Results The results showed that UBE2S is highly expressed in colorectal cancer and has a certain correlation with immune infiltrating cells. UBE2S promotes the proliferation and radiotherapy resistance of colorectal cancer cells; meanwhile, overexpression of UBE2S can activate the PI3K/AKT pathway, and this effect can be reversed by the addition of LY294002. In conclusion, our findings suggest that UBE2S may promote the proliferation and radiotherapy resistance of colorectal cancer cells by activating the PI3K/AKT signaling pathway. Conclusion In this study, we believe that UBE2S acts as an oncogene in colorectal cancer and can promote the progression of colorectal cancer by activating the PI3K/AKT pathway. The study of UBE2S not only contributes to a deeper understanding of the molecular mechanisms of colorectal cancer but also provides potential targets and strategies for clinical treatment. Intervention targeting UBE2S may improve the prognosis and treatment outcomes of colorectal cancer patients. UBE2S colorectal cancer PI3K/AKT proliferation Radiosensitivity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Colorectal cancer is the third most common malignant tumor worldwide, accounting for approximately 10% of all newly diagnosed cases of malignant tumors globally each year [ 1 ] . With the improvement of China's economic level, there have been significant changes in people's lifestyles and dietary habits. In recent years, the incidence and mortality rates of colorectal cancer in China have shown an upward trend [ 2 ] . Although certain progress has been made in the diagnosis and treatment of colorectal cancer in recent years, the prognosis of patients with advanced - stage disease remains poor, with a 5 - year survival rate of less than 15%. Therefore, it is of great significance to thoroughly elucidate the molecular mechanisms underlying the occurrence and development of colorectal cancer and to identify new therapeutic targets for improving the diagnosis and treatment of colorectal cancer. UBE2S, a member of the ubiquitin-conjugating enzyme E2 family, plays a crucial role in cell cycle regulation, DNA damage repair, and other cellular processes [ 3 ] . In recent years, numerous studies have demonstrated that UBE2S is upregulated in various malignant tumors and is believed to promote cancer cell proliferation, inhibit apoptosis, and contribute to the development and progression of multiple cancers [ 4 – 7 ] . For instance, in ovarian cancer, UBE2S promotes tumor cell proliferation and olaparib resistance by regulating the β-catenin signaling pathway [ 8 ] . In liver cancer, it suppresses tumor cell apoptosis through modulation of the p53 signaling pathway [ 9 ] . As a ubiquitin-conjugating enzyme E2, UBE2S can also influence the biological behavior and progression of malignancies via ubiquitination pathways. For example, it promotes lymph node metastasis in bladder cancer by interacting with TRIM21 to ubiquitinate and degrade LPP [ 10 ] . Additionally, it targets RPL26 for ubiquitination and degradation, thereby regulating c-Myc to drive the progression of non-small cell lung cancer [ 11 ] . However, the expression pattern, clinical significance, functional roles, and underlying mechanisms of UBE2S in colorectal cancer remain incompletely understood. PI3K is a family of heterodimeric lipid kinases, which are classified into classes I, II, and III subtypes. The class IA sub - group of PI3K activated by receptor tyrosine kinases consists of a p110 catalytic subunit and five p85 - like regulatory subunits. The class IB sub - group is composed of a catalytic subunit and a regulatory subunit. Class II PI3K includes PI3K - C2α (PIK3C2A), β (PIK3C2B), and γ (PIK3C2G). Class III PI3K is PIK3C3. When PI3K is activated by a variety of upstream cell - surface receptors, it catalyzes the generation of PIP3 from PIP2. PIP3 binds to the PH domain of AKT, promoting the recruitment of AKT to the cell membrane, where it is phosphorylated and activated by PDK1 and mTORC2. Activated AKT regulates processes such as cell proliferation, survival, and metabolism by phosphorylating downstream target proteins, such as mTOR, GSK − 3β, and FOXO [ 12 ] . The PI3K/AKT signaling pathway is an important signaling pathway that regulates cell proliferation, survival, and metabolism, and its abnormal activation is closely related to the development and progression of various tumors [ 13 ] . Current research has found that multiple oncogenes can promote the progression of cancer cells and resistance to treatment by activating the PI3K/AKT signaling pathway [ 14 – 15 ] . Therefore, blocking the activity of this pathway with targeted inhibitors could be a strategy for cancer treatment. This study aimed to investigate the expression of UBE2S in colorectal cancer and its impact on the proliferative capacity of colorectal cancer cells. Meanwhile, we explored the relationship between UBE2S and the PI3K/AKT signaling pathway. It was confirmed that UBE2S promoted the progression of colorectal cancer cells by activating the PI3K/AKT signaling pathway. This research will provide new theoretical basis for clarifying the pathogenesis of colorectal cancer and offer new potential targets for the targeted therapy of colorectal cancer. 2. Methods 2.1 Data downloading and sorting Download the gene expression datasets GSE110223, GSE110224, and GSE113513 from the Gene Expression Omnibus (GEO) database ( http://www.ncbi.nlm.nih.gov/geo ). Perform quality control, standardization, and normalization on the gene expression data within these datasets to obtain a gene expression matrix. Identify differentially expressed genes between colorectal cancer tissues and normal colorectal tissues using Perl and the "Limma" package in R. Screen for colorectal cancer characteristic genes using LASSO regression and SVM-RFE. LASSO regression is a regularized linear regression method that compresses and selects feature coefficients by adding an L1 norm penalty term to the loss function. SVM-RFE is a recursive feature elimination algorithm based on support vector machines (SVM), which constructs an optimal feature subset by iteratively removing the least important features. Download the transcriptome data of colorectal cancer and normal colorectal tissues from the TCGA database, and analyze the expression of UBE2S and its immune infiltration status in the TCGA database. Meanwhile, download the expression data of key genes in colorectal cancer tissues and normal colorectal tissues, as well as the relationship between such expression and patient prognosis, from the GEPIA database. 2.2 Cell Culture Human colorectal cancer cell lines RKO, Caco2, HT − 29 cells, and normal colorectal epithelial cells NCM460 were provided by the Translational Medicine Center of Yan'an University. RKO, HT − 29, and NCM460 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin in a cell incubator at 37°C with 5% CO₂. Caco2 cells were cultured in DMEM medium supplemented with 20% fetal bovine serum and 1% penicillin/streptomycin in a cell incubator at 37°C with 5% CO₂.The cell lines used in this study were all purchased from Servicebio, and no mycoplasma pollution occurred. 2.3 Construction of Stable Cell Lines with UBE2S Knockdown and Overexpression RKO and Caco2 cells in the logarithmic growth phase were selected. One day before infection, the cells were seeded into 6 - well plates to reach a confluence of 30% − 50% at the time of infection. On the next day, lentiviruses (purchased from Genechem) were transfected according to the method provided by the manufacturer. After 24–48 hours of infection, the culture medium was replaced with fresh medium. Twenty - four to forty - eight hours after medium replacement, medium containing puromycin was added, and the cells were continuously cultured for 1–2 weeks. During this period, non - infected cells would gradually die, while cells that had stably integrated the lentiviral vector would survive. The surviving cells were expanded, and the expression of UBE2S was verified by Western blot. 2.4 Western Blot Total proteins from cells and tissues were extracted using RIPA Buffer. Proteins were separated by 10% or 8% SDS - polyacrylamide gel electrophoresis and then transferred onto PVDF membranes. Subsequently, the membranes were blocked with 5% non - fat milk at room temperature for 2 hours. The membranes were then incubated with the primary antibody overnight at 4°C. After that, the membranes were incubated with the secondary antibody at room temperature for 2 hours, followed by washing three times with TBST. According to the manufacturer's instructions, the bands were visualized using an ECL detection system. 2.5 CCK8 Assay Approximately 1000 RKO and Caco2 cells were seeded into 96 - well plates and cultured for 1 day, 2 days, 3 days, 4 days, and 5 days respectively before terminating the culture. According to the manufacturer's instructions, 10 µL of Cell Counting Kit − 8 was added to each well, and the plates were then incubated in the incubator for 2 h. The optical density (OD) at 450 nm was measured using a microplate reader. 2.6 Colony Formation Assay A total of 500 cells in the logarithmic growth phase were seeded into 6 - well plates. After incubation for 1–2 weeks, the colonies were fixed with 4% paraformaldehyde for 30 minutes and then stained with 0.1% crystal violet solution for 15 minutes. The colony formation was observed. 2.7 Statistical Analysis All the above experiments were performed at least three times. Statistical analysis was carried out using Student's t - test in GraphPad Prism. P < 0.05 was considered statistically significant. 3. Results 3.1. UBE2S is highly expressed in colorectal cancer By extracting and analyzing data from three GEO datasets (GSE110223, GSE110224, and GSE113513) available on the GEO website ( https://www.ncbi.nlm.nih.gov/geo/ ) and combining them with colorectal cancer data from the TCGA database, we performed differential gene expression analysis. The results revealed a total of 175 differentially expressed genes (Fig. 1 A). These differentially expressed genes were subsequently subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses (Fig. 1 B, 1 C). Furthermore, we employed LASSO regression and SVM-RFE algorithms (Fig. 1 D, 1 E) to identify key genes, and the intersection of genes obtained from both methods yielded six differentially expressed genes: UBE2S, CHGA, PHLPP2, CHP2, CDH3, and TNFRSF17 (Fig. 1 F). 3.2 Correlative Analysis of UBE2S in Pan-Cancer Through literature review and pan-cancer analysis of data from The Cancer Genome Atlas (TCGA) database, we found that UBE2S is highly expressed in a variety of malignant tumors (Fig. 2 A). Moreover, the high expression of UBE2S exhibits a certain correlation with immune infiltrating cells across pan-cancer (Fig. 2 B). In addition, we further analyzed the relationship between UBE2S expression and patients’ Overall Survival (OS) as well as Disease-Specific Survival (DSS) in pan-cancer. The results revealed that UBE2S expression was correlated with OS in Adrenocortical Carcinoma (ACC), Diffuse Large B-Cell Lymphoma (DLBCL), Kidney Renal Clear Cell Carcinoma (KIRC), Brain Lower Grade Glioma (LGG), Liver Hepatocellular Carcinoma (LIHC), Lung Adenocarcinoma (LUAD), Mesothelioma (MESO), Skin Cutaneous Melanoma (SKCM), Uterine Corpus Endometrial Carcinoma (UCEC), and Uveal Melanoma (UVM) (Fig. 2 C). Meanwhile, UBE2S expression showed a correlation with DSS in ACC, KIRC, Kidney Renal Papillary Cell Carcinoma (KIRP), LGG, LIHC, LUAD, MESO, Rectum Adenocarcinoma (READ), SKCM, UCEC, and UVM (Fig. 2 D). In conclusion, we propose that UBE2S may be a meaningful gene and could play a certain role in tumor progression. 3.3 Analysis of UBE2S Expression in Colorectal Cancer and Its Correlation with Immune Cell Infiltration To further investigate the relationship between UBE2S and colorectal cancer, we analyzed the expression of UBE2S in colorectal cancer versus normal colorectal epithelium using the GEPIA database. We found that UBE2S was significantly highly expressed in colorectal cancer tissues (Fig. 3 A). Similarly, we downloaded data from The Cancer Genome Atlas (TCGA) database and analyzed UBE2S expression in colorectal cancer, which also revealed high expression of UBE2S in colorectal cancer (Fig. 3 B). Consistent results were obtained in paired colorectal cancer samples (Fig. 3 C). Western blot analysis was performed to detect UBE2S expression in colorectal cancer cells, and the results were consistent with our findings from public database analyses, further confirming the high expression of UBE2S in colorectal cancer (Fig. 3 D). Additionally, we analyzed the correlation between UBE2S and immune infiltrating cells in colorectal cancer, which showed that UBE2S was positively correlated with Th2 cells, NK CD56bright cells, NK CD56dim cells, aDC, CD8 T cells, and Cytotoxic cells, while negatively correlated with TFH, iDC, Eosinophils, Mast cells, Macrophages, pDC, Tem, T helper cells, and Tcm cells (Fig. 3 E). 3.4 Knockdown of UBE2S inhibits the proliferation of colorectal cancer cells To further explore the effect of UBE2S on the biological functions of colorectal cancer cells, we constructed a UBE2S low-expression colorectal cancer cell line (sh-UBE2S) and a negative control cell line (NC) via lentivirus transfection. Western blot was used to verify the changes in UBE2S protein level after UBE2S knockdown (Fig. 4 A). The results confirmed that the expression level of UBE2S in sh-UBE2S was lower than that in NC, demonstrating the successful construction of the sh-UBE2S cell line, which was then used in subsequent experiments. Next, colony formation assay confirmed that the proliferation ability of sh-UBE2S cells was significantly lower than that of NC cells (Fig. 4 B). The same result was obtained in the CCK-8 assay (Fig. 4 C). 3.5 Overexpression of UBE2S promotes the proliferation of colorectal cancer cells To further clarify the function of UBE2S in colorectal cancer cells, we simultaneously constructed a UBE2S-overexpressing colorectal cancer cell line (OE-UBE2S) and a negative control cell line (Vector) using lentivirus. We then further evaluated changes in the proliferation of OE-UBE2S and Vector cells. First, Western blot was similarly used to verify the changes in UBE2S protein level after UBE2S overexpression (Fig. 5 A). The results confirmed that the expression level of UBE2S in OE-UBE2S was higher than that in Vector, demonstrating the successful construction of the OE-UBE2S cell line, which could be used in subsequent experiments. Next, colony formation assay confirmed that the proliferation ability of OE-UBE2S cells was significantly stronger than that of Vector cells (Fig. 5 B). The same result was obtained in the CCK-8 assay (Fig. 5 C). 3.6 UBE2S Promotes Radiotherapy Resistance in Colorectal Cancer Cells Radiotherapy is one of the treatment modalities for colorectal cancer. To further investigate whether the high expression of UBE2S is associated with the sensitivity of colorectal cancer to radiotherapy, we cultured RKO and Caco2 cells in vitro and then treated these cells with different radiation doses (0 Gy, 2 Gy, 4 Gy, 6 Gy, and 8 Gy). Proteins were extracted 24 hours after irradiation, and Western blotting was used to detect the expression level of UBE2S in RKO and Caco2 cells. The results showed that the expression level of UBE2S increased with the increase in radiation dose (Fig. 6 A), demonstrating that radiotherapy can induce the expression of UBE2S. Western blotting was also used to detect the change in γ-H2AX after radiotherapy. It was found that after radiotherapy, the expression of γ-H2AX in the UBE2S-knockdown group was higher than that in the NC group, while the expression of γ-H2AX in the UBE2S-overexpression group was lower than that in the Vector group (Fig. 6 B). These results confirm that knockdown of UBE2S leads to more severe cell damage caused by radiotherapy, and UBE2S knockdown can increase the sensitivity of cancer cells to radiotherapy. 3.7 RNA - Seq analysis indicates that UBE2S is enriched in the PI3K/AKT pathway The above experimental results confirm that UBE2S functions as an oncogene in colorectal cancer. UBE2S can enhance the proliferative capacity of colorectal cancer cells and inhibit the level of apoptosis. To further elucidate its specific mechanism of action, we analyzed the differentially expressed genes between the sh - UBE2S and NC groups by RNA - Seq (Fig. 7 A, Fig. 7 B). The results showed that UBE2S was enriched in pathways such as the cell cycle, apoptosis, PI3K/AKT pathway, DNA replication, base excision repair, ferroptosis, and non - homologous end - joining (Fig. 7 C). Among these, the PI3K/AKT pathway is the most significant pathway for the biological behavior of cancer cells. We performed PPI network analysis on the genes enriched in the PI3K/AKT pathway(Figure 7 D). Therefore, we speculate that UBE2S may promote the proliferative capacity of colorectal cancer cells by regulating the PI3K/AKT pathway. 3.5 UBE2S promotes the proliferation of colorectal cancer cells by regulating the PI3K/AKT signaling pathway The sequencing results suggest that UBE2S is enriched in the PI3K/AKT signaling pathway. To further verify whether UBE2S can regulate the PI3K/AKT signaling pathway to enhance the proliferation of colorectal cancer cells and inhibit their apoptosis, we detected the protein expression levels of p - PI3K, PI3K, p - AKT, and AKT in sh - UBE2S and NC cells, as well as in OE - UBE2S and Vector cells by western blot. We found that after knocking down UBE2S, the expression levels of p - PI3K and p - AKT were significantly decreased, while the changes in PI3K and AKT were not significant. In contrast, after overexpressing UBE2S, the expression levels of p - PI3K and p - AKT were significantly increased, with no significant changes in PI3K and AKT (Fig. 8 A). To further clarify whether UBE2S can regulate the PI3K/AKT signaling pathway, we added the PI3K/AKT signaling pathway inhibitor LY294002 to OE - UBE2S and Vector cells, and then detected the protein expression levels of p - PI3K, PI3K, p - AKT, and AKT by western blot again. The results showed that after adding LY294002, the expression levels of p - PI3K and p - AKT were significantly decreased, while the changes in PI3K and AKT were not significant. Notably, the expression levels of p - PI3K and p - AKT in OE - UBE2S cells were still higher than those in the Vector group after adding the pathway inhibitor (Fig. 8 B). The CCK8 assay further verified that UBE2S can enhance the proliferation ability of colorectal cancer cells by regulating the PI3K/AKT signaling pathway (Fig. 8 C). 4. Discussion Colorectal cancer remains one of the most prevalent malignant tumors of the digestive tract worldwide in terms of both incidence and mortality rates. Epidemiologic studies reveal significant geographic disparities, with developed nations in Europe demonstrating higher incidence rates compared to developing regions in Asia and Africa. In recent years, a concerning epidemiological shift has been observed as developing countries progressively adopt Westernized dietary patterns and lifestyle habits similar to those prevalent in Europe and North America. This nutritional transition has been correlated with a steady increase in colorectal cancer incidence across developing nations [ 16 ] .Diet, lifestyle, and genetic factors play significant roles in the development and progression of colorectal cancer. Obesity, alcohol consumption, and diets high in sugar and fat are strongly associated with an increased incidence of colorectal cancer. Furthermore, research by Yang et al. demonstrated that high-fat diets can promote the development of colorectal cancer [ 17 ] ,Higher intake of sugar-sweetened beverages during adulthood and adolescence is associated with an increased risk of early-onset colorectal cancer in women [ 18 ] .Numerous studies have established that diets high in sugar and fat are strongly associated with an elevated risk of colorectal cancer. Additionally, genetic predisposition constitutes a significant risk factor for this malignancy. Hereditary syndromes such as Lynch syndrome and familial adenomatous polyposis (FAP) have been shown to substantially increase colorectal cancer susceptibility.Lynch syndrome, the most prevalent inherited colorectal cancer syndrome, accounts for approximately 3% of all colorectal cancer cases. This condition is characterized by mismatch repair gene mutations, which lead to microsatellite instability and subsequent cancer predisposition. FAP, caused by APC gene mutations, manifests as the development of hundreds of adenomatous polyps throughout the colon and rectum, with near-certain progression to colorectal cancer if left untreated [ 19 ] . Ubiquitination, a crucial post-translational modification in maintaining cellular protein homeostasis, is mediated by a three-enzyme cascade: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3) [ 20 ] .Emerging evidence has highlighted the critical involvement of ubiquitination in tumorigenesis and cancer progression. For instance, studies by Li et al. have demonstrated that the E3 ligase CUL3 interacts with BECN1, mediating the degradation of Beclin-1. This process suppresses autophagy in breast cancer cells, thereby promoting tumor progression [ 21 ] .UBE2S, a member of the ubiquitin-conjugating enzyme (E2) family, exhibits oncogenic functions across multiple tumor types. By interacting with ubiquitin ligase TRIM21, UBE2S mediates K11-linked ubiquitination of LPP, thereby promoting lymph node metastasis in bladder cancer [ 22 – 27 ] .Concurrently, studies have demonstrated that UBE2S promotes the progression of ovarian cancer. However, its precise functional roles and underlying mechanisms in colorectal cancer remain incompletely elucidated. The PI3K/AKT signaling pathway plays a pivotal regulatory role across diverse cell types. In malignant cells, this pathway is frequently aberrantly activated, thereby driving hyperproliferation through sustained oncogenic signaling [ 28 ] .Activation of PI3K phosphorylates AKT, triggering its translocation from the plasma membrane to the cytoplasm. This process subsequently activates downstream signaling cascades that promote cell survival and proliferation. The aberrant activation of the PI3K/AKT signaling pathway exerts profound effects on the malignant phenotypes of cancer cells. Emerging evidence has established that specific molecules drive tumorigenesis and progression through modulation of this pathway [ 29 ] . Notably, Xu et al. demonstrated that NETO2 regulates the PI3K/AKT axis to induce proliferation and metastatic dissemination in esophageal carcinoma cells [ 30 ] .Studies by Haines et al. have revealed that GREB1 modulates the PI3K/AKT signaling pathway to regulate proliferation in hormone-sensitive breast cancer [ 31 ] . In colorectal cancer, aberrant activation of the PI3K/AKT signaling pathway is closely associated with tumorigenesis, progression, and chemotherapy resistance. Inhibitors targeting this pathway have emerged as a promising therapeutic strategy [ 32 ] . Additionally, multiple genes have been demonstrated to influence colorectal cancer progression and treatment resistance through regulation of the PI3K/AKT signaling pathway. For instance, Claudin14 promotes colorectal cancer progression by activating the PI3K/AKT/mTOR pathway [ 33 ] . Similarly, BEST4 exerts its oncogenic function in colorectal cancer via activation of the PI3K/Akt signaling pathway [ 34 ] . Other molecules such as eIf3a, MUC3A, and LIN28B have also been confirmed to mediate malignant biological behaviors in colorectal cancer through modulation of the PI3K/AKT pathway [ 35 – 37 ] . Therefore, we conclude that activation of the PI3K/AKT signaling pathway is critically involved in the disease progression of colorectal cancer. First, this study utilized GEO and TCGA datasets to conduct bioinformatics analysis on differentially expressed genes between normal colorectal tissues and colorectal cancer tissues. Further, LASSO and SVM-RFE methods were employed to identify characteristic genes of colorectal cancer, and UBE2S was found to be a characteristic gene of this malignancy. Through pan-cancer analysis of data from The Cancer Genome Atlas (TCGA) database, we observed that UBE2S is highly expressed in a variety of malignant tumors. In addition, the high expression of UBE2S showed a certain correlation with immune-infiltrating cells across pan-cancers. Therefore, we consider UBE2S to be a biologically meaningful gene that may play a role in tumor progression. Analysis of the GEPIA and TCGA databases revealed that UBE2S is differentially overexpressed in colorectal cancer and is associated with cellular immune infiltration. Subsequently, the expression of UBE2S in colorectal cancer was verified using Western blot. To clarify the significance of the differential expression of UBE2S, our experimental investigations demonstrated that knockdown of UBE2S inhibited the proliferation of colorectal cancer cells and increased their sensitivity to radiotherapy, whereas overexpression of UBE2S enhanced the proliferative capacity of cancer cells and reduced their sensitivity to radiotherapy. Furthermore, RNA sequencing analysis showed that UBE2S was significantly enriched in the PI3K/AKT pathway. We thus further explored the relationship between UBE2S and the PI3K/AKT signaling pathway, and the results indicated that UBE2S may promote the progression of colorectal cancer cells and their resistance to radiotherapy by activating the classical PI3K/AKT signaling pathway. This study confirms that UBE2S can enhance the proliferative capacity and radiotherapy resistance of colorectal cancer cells by activating the PI3K/AKT signaling pathway, though the specific mechanism underlying this process has not been explored in depth. Future research will focus on the specific manner in which UBE2S activates the PI3K/AKT signaling pathway. Based on the findings of this study, given that UBE2S functions as an oncogene in colorectal cancer, small-molecule inhibitors targeting UBE2S are expected to become a novel therapeutic option for patients with colorectal cancer. 5. Conclusion In conclusion, we believe that UBE2S functions as an oncogene in colorectal cancer and can promote the progression and radiotherapy resistance of colorectal cancer by activating the PI3K/AKT pathway. Research on UBE2S not only helps to gain a deeper understanding of the molecular mechanisms underlying colorectal cancer but also provides potential targets and strategies for clinical treatment. Interventions targeting UBE2S may improve the prognosis and treatment outcomes of patients with colorectal cancer. Declarations Author Contributions Yu Chang and Yufeng Hu designed and performed the experiments. Jing Guo and Yaqin Hao analyzed the experimental data. Fei Huang and Yu Liu wrote the paper. Yu Chang and Kai Bai revised the manuscript and submitted it for publication. Funding This study was supported by Construction of Healthy Gastric Cancer Organs and Innovative Team of Clinical Application Research in Shaanxi Province[ 2025TD-20]. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Confilict of interest statement The authors declare no competing interests. Data Availability All data generated or analysed during this study are included in this published article and its supplementary information files. References Klimeck L, Heisser T, Hoffmeister M, Brenner H. Colorectal cancer: A health and economic problem. Best Pract Res Clin Gastroenterol. 2023;66:101839. Li N, Lu B, Luo C, et al. Incidence, mortality, survival, risk factor and screening of colorectal cancer: A comparison among China, Europe, and northern America. Cancer Lett. 2021;522:255–68. Wang L, Liang Y, Li P, Liang Q, Sun H, Xu D, Hu W. Oncogenic Activities Of UBE2S Mediated By VHL/HIF-1α/STAT3 Signal Via The Ubiquitin-Proteasome System In PDAC. Onco Targets Ther. 2019;12:9767–81. 10.2147/OTT.S228522 . Zhang M, Liu Y, Yin Y, et al. 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Huo C, Wu D, Li X, Zhang Y, Hu B, Zhang T, Ren J, Wang T, Liu Y. eIf3a mediates malignant biological behaviors in colorectal cancer through the PI3K/AKT signaling pathway. Cancer Biol Ther. 2024;25(1):2355703. Su W, Feng B, Hu L, Guo X, Yu M. MUC3A promotes the progression of colorectal cancer through the PI3K/Akt/mTOR pathway. BMC Cancer. 2022;22(1):602. Shin AE, Sugiura K, Kariuki SW, Cohen DA, Flashner SP, Klein-Szanto AJ, Nishiwaki N, De D, Vasan N, Gabre JT, Lengner CJ, Sims PA, Rustgi AK. LIN28B-mediated PI3K/AKT pathway activation promotes metastasis in colorectal cancer models. J Clin Invest. 2025;135(8):e186035. Additional Declarations No competing interests reported. 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7724776","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":534728673,"identity":"f3edc439-7207-4676-95d7-5dd9ed1659da","order_by":0,"name":"Yu Chang","email":"","orcid":"","institution":"The First Affiliated Hospital of Yan’an University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Chang","suffix":""},{"id":534728674,"identity":"ce5b6f59-41a5-45a1-81c2-6965dd4dc57a","order_by":1,"name":"Yu Liu","email":"","orcid":"","institution":"The First Affiliated Hospital of Yan’an 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1","display":"","copyAsset":false,"role":"figure","size":257763,"visible":true,"origin":"","legend":"\u003cp\u003eUBE2S is highly expressed in colorectal cancer. (A) Differentially expressed genes from the intersection of the GEO database and the TCGA database. (B, C) GO and KEGG enrichment analyses of the differentially expressed genes. (D, E, F) Differentially expressed genes with a high correlation to colorectal cancer were obtained through LASSO regression and SVM - RFE.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7724776/v1/e92ace3d2d183c31d09ada86.png"},{"id":94697462,"identity":"9cf249d6-326d-4e9d-b7c1-dd8a5338b165","added_by":"auto","created_at":"2025-10-29 18:37:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":417954,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelative analysis of UBE2S in pan-cancer. (A)Expression of UBE2S in pan-cancer;(B) Correlation analysis between UBE2S and immune infiltrating cells in pan-cancer;(C) Correlation between UBE2S expression and patients’ OS in pan-cancer;(D) Correlation between UBE2S expression and patients’ DSS in pan-cancer.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7724776/v1/130b06222fb247019a2780c0.png"},{"id":94697464,"identity":"ecf69bb8-dad6-4345-8f23-1e6bc1b80cb3","added_by":"auto","created_at":"2025-10-29 18:37:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":314614,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of UBE2S expression in colorectal cancer and its correlation with immune cell infiltration.(A) GEPIA database showing high expression of UBE2S in colorectal cancer;(B) TCGA database showing high expression of UBE2S in colorectal cancer;(C) Paired samples from TCGA database showing high expression of UBE2S in colorectal cancer;(D) Western blot results showing high expression of UBE2S in colorectal cancer;(E) Correlation analysis between UBE2S expression and immune infiltrating cells in colorectal cancer.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7724776/v1/bb85907c05ea0436834cdb5b.png"},{"id":94728408,"identity":"f85545aa-1bf9-41a8-9782-7ade19ec7333","added_by":"auto","created_at":"2025-10-30 07:03:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":214625,"visible":true,"origin":"","legend":"\u003cp\u003eKnockdown of UBE2S Inhibits the Proliferation of Colorectal Cancer Cells (A) Establishment of stable colorectal cancer cell lines with UBE2S knockdown. (B) Colony formation assay verified that knockdown of UBE2S inhibits the proliferation of colorectal cancer cells. (C,D) CCK8 assay was used to detect that knockdown of UBE2S inhibits the proliferation of colorectal cancer cells.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7724776/v1/b4c06c7be64e3a77370daf1f.png"},{"id":94697469,"identity":"7d979507-6e32-4ce5-b049-52006dde4c4f","added_by":"auto","created_at":"2025-10-29 18:37:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":237838,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of UBE2S Promotes the Proliferation of Colorectal Cancer Cells (A) Construction of stable colorectal cancer cell lines with overexpressed UBE2S. (B) The colony - formation assay verified that overexpression of UBE2S promotes the proliferation of colorectal cancer cells.(C,D) The CCK8 assay was used to detect that overexpression of UBE2S promotes the proliferation of colorectal cancer cells.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7724776/v1/ce6a5fff46ba40f1c16e6eb4.png"},{"id":94728253,"identity":"c60a8cdb-1597-440a-b401-81446f3bd639","added_by":"auto","created_at":"2025-10-30 07:03:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":252666,"visible":true,"origin":"","legend":"\u003cp\u003eUBE2S promotes radiotherapy resistance in colorectal cancer cells.(A) Radiation significantly induces the expression of UBE2S;(B) Knockdown of UBE2S enhances the sensitivity of colorectal cancer cells to radiation.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7724776/v1/f1de1d3850dd90683167cf83.png"},{"id":94697471,"identity":"46f5f920-31f6-46d8-99d4-2bfbe830eabd","added_by":"auto","created_at":"2025-10-29 18:37:20","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":354794,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The volcano plot shows the differentially expressed genes between sh - UBE2S and NC cells. (B) The heatmap shows the differentially expressed genes between sh - UBE2S and NC cells. (C) The related pathways enriched by UBE2S. (D) The PPI network of molecules in the PI3K/AKT signaling pathway.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7724776/v1/b4705a3c8038667b7df5282b.png"},{"id":94697478,"identity":"e64f4abd-f4ec-4b57-8324-e16aef5199db","added_by":"auto","created_at":"2025-10-29 18:37:20","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":521866,"visible":true,"origin":"","legend":"\u003cp\u003eUBE2S promotes the proliferation of colorectal cancer cells by regulating the PI3K/AKT signaling pathway. (A) UBE2S activates the PI3K/AKT signaling pathway. (B) The addition of a pathway inhibitor suppresses the activation of the PI3K/AKT signaling pathway by UBE2S. (C) The CCK8 assay confirms that UBE2S promotes the proliferation of colorectal cancer cells by activating the PI3K/AKT signaling pathway.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7724776/v1/51df5a5186286c5a53b65837.png"},{"id":104398577,"identity":"7d6b8ed0-af7a-4305-8763-e5ad27ce964f","added_by":"auto","created_at":"2026-03-11 12:03:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3207635,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7724776/v1/6f545cbf-3ff6-4082-8881-a7a374e74139.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"From Data Mining to Functional Validation: UBE2S Regulates Proliferation and Radiosensitivity in Colorectal Cancer via the PI3K/AKT Pathway","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eColorectal cancer is the third most common malignant tumor worldwide, accounting for approximately 10% of all newly diagnosed cases of malignant tumors globally each year \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. With the improvement of China's economic level, there have been significant changes in people's lifestyles and dietary habits. In recent years, the incidence and mortality rates of colorectal cancer in China have shown an upward trend \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Although certain progress has been made in the diagnosis and treatment of colorectal cancer in recent years, the prognosis of patients with advanced - stage disease remains poor, with a 5 - year survival rate of less than 15%. Therefore, it is of great significance to thoroughly elucidate the molecular mechanisms underlying the occurrence and development of colorectal cancer and to identify new therapeutic targets for improving the diagnosis and treatment of colorectal cancer.\u003c/p\u003e\u003cp\u003eUBE2S, a member of the ubiquitin-conjugating enzyme E2 family, plays a crucial role in cell cycle regulation, DNA damage repair, and other cellular processes \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. In recent years, numerous studies have demonstrated that UBE2S is upregulated in various malignant tumors and is believed to promote cancer cell proliferation, inhibit apoptosis, and contribute to the development and progression of multiple cancers \u003csup\u003e[\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. For instance, in ovarian cancer, UBE2S promotes tumor cell proliferation and olaparib resistance by regulating the β-catenin signaling pathway \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. In liver cancer, it suppresses tumor cell apoptosis through modulation of the p53 signaling pathway \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. As a ubiquitin-conjugating enzyme E2, UBE2S can also influence the biological behavior and progression of malignancies via ubiquitination pathways. For example, it promotes lymph node metastasis in bladder cancer by interacting with TRIM21 to ubiquitinate and degrade LPP \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Additionally, it targets RPL26 for ubiquitination and degradation, thereby regulating c-Myc to drive the progression of non-small cell lung cancer \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. However, the expression pattern, clinical significance, functional roles, and underlying mechanisms of UBE2S in colorectal cancer remain incompletely understood.\u003c/p\u003e\u003cp\u003ePI3K is a family of heterodimeric lipid kinases, which are classified into classes I, II, and III subtypes. The class IA sub - group of PI3K activated by receptor tyrosine kinases consists of a p110 catalytic subunit and five p85 - like regulatory subunits. The class IB sub - group is composed of a catalytic subunit and a regulatory subunit. Class II PI3K includes PI3K - C2α (PIK3C2A), β (PIK3C2B), and γ (PIK3C2G). Class III PI3K is PIK3C3. When PI3K is activated by a variety of upstream cell - surface receptors, it catalyzes the generation of PIP3 from PIP2. PIP3 binds to the PH domain of AKT, promoting the recruitment of AKT to the cell membrane, where it is phosphorylated and activated by PDK1 and mTORC2. Activated AKT regulates processes such as cell proliferation, survival, and metabolism by phosphorylating downstream target proteins, such as mTOR, GSK \u0026minus;\u0026thinsp;3β, and FOXO \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. The PI3K/AKT signaling pathway is an important signaling pathway that regulates cell proliferation, survival, and metabolism, and its abnormal activation is closely related to the development and progression of various tumors \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Current research has found that multiple oncogenes can promote the progression of cancer cells and resistance to treatment by activating the PI3K/AKT signaling pathway \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e \u0026ndash; \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Therefore, blocking the activity of this pathway with targeted inhibitors could be a strategy for cancer treatment.\u003c/p\u003e\u003cp\u003eThis study aimed to investigate the expression of UBE2S in colorectal cancer and its impact on the proliferative capacity of colorectal cancer cells. Meanwhile, we explored the relationship between UBE2S and the PI3K/AKT signaling pathway. It was confirmed that UBE2S promoted the progression of colorectal cancer cells by activating the PI3K/AKT signaling pathway. This research will provide new theoretical basis for clarifying the pathogenesis of colorectal cancer and offer new potential targets for the targeted therapy of colorectal cancer.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Data downloading and sorting\u003c/h2\u003e\u003cp\u003eDownload the gene expression datasets GSE110223, GSE110224, and GSE113513 from the Gene Expression Omnibus (GEO) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/geo\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/geo\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Perform quality control, standardization, and normalization on the gene expression data within these datasets to obtain a gene expression matrix. Identify differentially expressed genes between colorectal cancer tissues and normal colorectal tissues using Perl and the \"Limma\" package in R. Screen for colorectal cancer characteristic genes using LASSO regression and SVM-RFE. LASSO regression is a regularized linear regression method that compresses and selects feature coefficients by adding an L1 norm penalty term to the loss function. SVM-RFE is a recursive feature elimination algorithm based on support vector machines (SVM), which constructs an optimal feature subset by iteratively removing the least important features. Download the transcriptome data of colorectal cancer and normal colorectal tissues from the TCGA database, and analyze the expression of UBE2S and its immune infiltration status in the TCGA database. Meanwhile, download the expression data of key genes in colorectal cancer tissues and normal colorectal tissues, as well as the relationship between such expression and patient prognosis, from the GEPIA database.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Cell Culture\u003c/h2\u003e\u003cp\u003eHuman colorectal cancer cell lines RKO, Caco2, HT \u0026minus;\u0026thinsp;29 cells, and normal colorectal epithelial cells NCM460 were provided by the Translational Medicine Center of Yan'an University. RKO, HT \u0026minus;\u0026thinsp;29, and NCM460 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin in a cell incubator at 37\u0026deg;C with 5% CO₂. Caco2 cells were cultured in DMEM medium supplemented with 20% fetal bovine serum and 1% penicillin/streptomycin in a cell incubator at 37\u0026deg;C with 5% CO₂.The cell lines used in this study were all purchased from Servicebio, and no mycoplasma pollution occurred.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Construction of Stable Cell Lines with UBE2S Knockdown and Overexpression\u003c/h2\u003e\u003cp\u003eRKO and Caco2 cells in the logarithmic growth phase were selected. One day before infection, the cells were seeded into 6 - well plates to reach a confluence of 30% \u0026minus;\u0026thinsp;50% at the time of infection. On the next day, lentiviruses (purchased from Genechem) were transfected according to the method provided by the manufacturer. After 24\u0026ndash;48 hours of infection, the culture medium was replaced with fresh medium. Twenty - four to forty - eight hours after medium replacement, medium containing puromycin was added, and the cells were continuously cultured for 1\u0026ndash;2 weeks. During this period, non - infected cells would gradually die, while cells that had stably integrated the lentiviral vector would survive. The surviving cells were expanded, and the expression of UBE2S was verified by Western blot.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Western Blot\u003c/h2\u003e\u003cp\u003eTotal proteins from cells and tissues were extracted using RIPA Buffer. Proteins were separated by 10% or 8% SDS - polyacrylamide gel electrophoresis and then transferred onto PVDF membranes. Subsequently, the membranes were blocked with 5% non - fat milk at room temperature for 2 hours. The membranes were then incubated with the primary antibody overnight at 4\u0026deg;C. After that, the membranes were incubated with the secondary antibody at room temperature for 2 hours, followed by washing three times with TBST. According to the manufacturer's instructions, the bands were visualized using an ECL detection system.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 CCK8 Assay\u003c/h2\u003e\u003cp\u003eApproximately 1000 RKO and Caco2 cells were seeded into 96 - well plates and cultured for 1 day, 2 days, 3 days, 4 days, and 5 days respectively before terminating the culture. According to the manufacturer's instructions, 10 \u0026micro;L of Cell Counting Kit \u0026minus;\u0026thinsp;8 was added to each well, and the plates were then incubated in the incubator for 2 h. The optical density (OD) at 450 nm was measured using a microplate reader.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Colony Formation Assay\u003c/h2\u003e\u003cp\u003eA total of 500 cells in the logarithmic growth phase were seeded into 6 - well plates. After incubation for 1\u0026ndash;2 weeks, the colonies were fixed with 4% paraformaldehyde for 30 minutes and then stained with 0.1% crystal violet solution for 15 minutes. The colony formation was observed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Statistical Analysis\u003c/h2\u003e\u003cp\u003eAll the above experiments were performed at least three times. Statistical analysis was carried out using Student's t - test in GraphPad Prism. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.1. UBE2S is highly expressed in colorectal cancer\u003c/h2\u003e\u003cp\u003eBy extracting and analyzing data from three GEO datasets (GSE110223, GSE110224, and GSE113513) available on the GEO website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/geo/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and combining them with colorectal cancer data from the TCGA database, we performed differential gene expression analysis. The results revealed a total of 175 differentially expressed genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). These differentially expressed genes were subsequently subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Furthermore, we employed LASSO regression and SVM-RFE algorithms (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) to identify key genes, and the intersection of genes obtained from both methods yielded six differentially expressed genes: UBE2S, CHGA, PHLPP2, CHP2, CDH3, and TNFRSF17 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Correlative Analysis of UBE2S in Pan-Cancer\u003c/h2\u003e\u003cp\u003eThrough literature review and pan-cancer analysis of data from The Cancer Genome Atlas (TCGA) database, we found that UBE2S is highly expressed in a variety of malignant tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Moreover, the high expression of UBE2S exhibits a certain correlation with immune infiltrating cells across pan-cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In addition, we further analyzed the relationship between UBE2S expression and patients\u0026rsquo; Overall Survival (OS) as well as Disease-Specific Survival (DSS) in pan-cancer. The results revealed that UBE2S expression was correlated with OS in Adrenocortical Carcinoma (ACC), Diffuse Large B-Cell Lymphoma (DLBCL), Kidney Renal Clear Cell Carcinoma (KIRC), Brain Lower Grade Glioma (LGG), Liver Hepatocellular Carcinoma (LIHC), Lung Adenocarcinoma (LUAD), Mesothelioma (MESO), Skin Cutaneous Melanoma (SKCM), Uterine Corpus Endometrial Carcinoma (UCEC), and Uveal Melanoma (UVM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Meanwhile, UBE2S expression showed a correlation with DSS in ACC, KIRC, Kidney Renal Papillary Cell Carcinoma (KIRP), LGG, LIHC, LUAD, MESO, Rectum Adenocarcinoma (READ), SKCM, UCEC, and UVM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). In conclusion, we propose that UBE2S may be a meaningful gene and could play a certain role in tumor progression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Analysis of UBE2S Expression in Colorectal Cancer and Its Correlation with Immune Cell Infiltration\u003c/h2\u003e\u003cp\u003eTo further investigate the relationship between UBE2S and colorectal cancer, we analyzed the expression of UBE2S in colorectal cancer versus normal colorectal epithelium using the GEPIA database. We found that UBE2S was significantly highly expressed in colorectal cancer tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Similarly, we downloaded data from The Cancer Genome Atlas (TCGA) database and analyzed UBE2S expression in colorectal cancer, which also revealed high expression of UBE2S in colorectal cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Consistent results were obtained in paired colorectal cancer samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Western blot analysis was performed to detect UBE2S expression in colorectal cancer cells, and the results were consistent with our findings from public database analyses, further confirming the high expression of UBE2S in colorectal cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Additionally, we analyzed the correlation between UBE2S and immune infiltrating cells in colorectal cancer, which showed that UBE2S was positively correlated with Th2 cells, NK CD56bright cells, NK CD56dim cells, aDC, CD8 T cells, and Cytotoxic cells, while negatively correlated with TFH, iDC, Eosinophils, Mast cells, Macrophages, pDC, Tem, T helper cells, and Tcm cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Knockdown of UBE2S inhibits the proliferation of colorectal cancer cells\u003c/h2\u003e\u003cp\u003eTo further explore the effect of UBE2S on the biological functions of colorectal cancer cells, we constructed a UBE2S low-expression colorectal cancer cell line (sh-UBE2S) and a negative control cell line (NC) via lentivirus transfection. Western blot was used to verify the changes in UBE2S protein level after UBE2S knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The results confirmed that the expression level of UBE2S in sh-UBE2S was lower than that in NC, demonstrating the successful construction of the sh-UBE2S cell line, which was then used in subsequent experiments. Next, colony formation assay confirmed that the proliferation ability of sh-UBE2S cells was significantly lower than that of NC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The same result was obtained in the CCK-8 assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e3.5 Overexpression of UBE2S promotes the proliferation of colorectal cancer cells\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eTo further clarify the function of UBE2S in colorectal cancer cells, we simultaneously constructed a UBE2S-overexpressing colorectal cancer cell line (OE-UBE2S) and a negative control cell line (Vector) using lentivirus. We then further evaluated changes in the proliferation of OE-UBE2S and Vector cells. First, Western blot was similarly used to verify the changes in UBE2S protein level after UBE2S overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The results confirmed that the expression level of UBE2S in OE-UBE2S was higher than that in Vector, demonstrating the successful construction of the OE-UBE2S cell line, which could be used in subsequent experiments. Next, colony formation assay confirmed that the proliferation ability of OE-UBE2S cells was significantly stronger than that of Vector cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The same result was obtained in the CCK-8 assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.6 UBE2S Promotes Radiotherapy Resistance in Colorectal Cancer Cells\u003c/h2\u003e\u003cp\u003eRadiotherapy is one of the treatment modalities for colorectal cancer. To further investigate whether the high expression of UBE2S is associated with the sensitivity of colorectal cancer to radiotherapy, we cultured RKO and Caco2 cells in vitro and then treated these cells with different radiation doses (0 Gy, 2 Gy, 4 Gy, 6 Gy, and 8 Gy). Proteins were extracted 24 hours after irradiation, and Western blotting was used to detect the expression level of UBE2S in RKO and Caco2 cells. The results showed that the expression level of UBE2S increased with the increase in radiation dose (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), demonstrating that radiotherapy can induce the expression of UBE2S. Western blotting was also used to detect the change in γ-H2AX after radiotherapy. It was found that after radiotherapy, the expression of γ-H2AX in the UBE2S-knockdown group was higher than that in the NC group, while the expression of γ-H2AX in the UBE2S-overexpression group was lower than that in the Vector group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). These results confirm that knockdown of UBE2S leads to more severe cell damage caused by radiotherapy, and UBE2S knockdown can increase the sensitivity of cancer cells to radiotherapy.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e3.7 RNA - Seq analysis indicates that UBE2S is enriched in the PI3K/AKT pathway\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eThe above experimental results confirm that UBE2S functions as an oncogene in colorectal cancer. UBE2S can enhance the proliferative capacity of colorectal cancer cells and inhibit the level of apoptosis. To further elucidate its specific mechanism of action, we analyzed the differentially expressed genes between the sh - UBE2S and NC groups by RNA - Seq (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). The results showed that UBE2S was enriched in pathways such as the cell cycle, apoptosis, PI3K/AKT pathway, DNA replication, base excision repair, ferroptosis, and non - homologous end - joining (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Among these, the PI3K/AKT pathway is the most significant pathway for the biological behavior of cancer cells. We performed PPI network analysis on the genes enriched in the PI3K/AKT pathway(Figure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Therefore, we speculate that UBE2S may promote the proliferative capacity of colorectal cancer cells by regulating the PI3K/AKT pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.5 UBE2S promotes the proliferation of colorectal cancer cells by regulating the PI3K/AKT signaling pathway\u003c/h2\u003e\u003cp\u003eThe sequencing results suggest that UBE2S is enriched in the PI3K/AKT signaling pathway. To further verify whether UBE2S can regulate the PI3K/AKT signaling pathway to enhance the proliferation of colorectal cancer cells and inhibit their apoptosis, we detected the protein expression levels of p - PI3K, PI3K, p - AKT, and AKT in sh - UBE2S and NC cells, as well as in OE - UBE2S and Vector cells by western blot. We found that after knocking down UBE2S, the expression levels of p - PI3K and p - AKT were significantly decreased, while the changes in PI3K and AKT were not significant. In contrast, after overexpressing UBE2S, the expression levels of p - PI3K and p - AKT were significantly increased, with no significant changes in PI3K and AKT (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). To further clarify whether UBE2S can regulate the PI3K/AKT signaling pathway, we added the PI3K/AKT signaling pathway inhibitor LY294002 to OE - UBE2S and Vector cells, and then detected the protein expression levels of p - PI3K, PI3K, p - AKT, and AKT by western blot again. The results showed that after adding LY294002, the expression levels of p - PI3K and p - AKT were significantly decreased, while the changes in PI3K and AKT were not significant. Notably, the expression levels of p - PI3K and p - AKT in OE - UBE2S cells were still higher than those in the Vector group after adding the pathway inhibitor (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). The CCK8 assay further verified that UBE2S can enhance the proliferation ability of colorectal cancer cells by regulating the PI3K/AKT signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eColorectal cancer remains one of the most prevalent malignant tumors of the digestive tract worldwide in terms of both incidence and mortality rates. Epidemiologic studies reveal significant geographic disparities, with developed nations in Europe demonstrating higher incidence rates compared to developing regions in Asia and Africa. In recent years, a concerning epidemiological shift has been observed as developing countries progressively adopt Westernized dietary patterns and lifestyle habits similar to those prevalent in Europe and North America. This nutritional transition has been correlated with a steady increase in colorectal cancer incidence across developing nations\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e.Diet, lifestyle, and genetic factors play significant roles in the development and progression of colorectal cancer. Obesity, alcohol consumption, and diets high in sugar and fat are strongly associated with an increased incidence of colorectal cancer. Furthermore, research by Yang et al. demonstrated that high-fat diets can promote the development of colorectal cancer\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e,Higher intake of sugar-sweetened beverages during adulthood and adolescence is associated with an increased risk of early-onset colorectal cancer in women\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e.Numerous studies have established that diets high in sugar and fat are strongly associated with an elevated risk of colorectal cancer. Additionally, genetic predisposition constitutes a significant risk factor for this malignancy. Hereditary syndromes such as Lynch syndrome and familial adenomatous polyposis (FAP) have been shown to substantially increase colorectal cancer susceptibility.Lynch syndrome, the most prevalent inherited colorectal cancer syndrome, accounts for approximately 3% of all colorectal cancer cases. This condition is characterized by mismatch repair gene mutations, which lead to microsatellite instability and subsequent cancer predisposition. FAP, caused by APC gene mutations, manifests as the development of hundreds of adenomatous polyps throughout the colon and rectum, with near-certain progression to colorectal cancer if left untreated\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eUbiquitination, a crucial post-translational modification in maintaining cellular protein homeostasis, is mediated by a three-enzyme cascade: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3)\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.Emerging evidence has highlighted the critical involvement of ubiquitination in tumorigenesis and cancer progression. For instance, studies by Li et al. have demonstrated that the E3 ligase CUL3 interacts with BECN1, mediating the degradation of Beclin-1. This process suppresses autophagy in breast cancer cells, thereby promoting tumor progression\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e.UBE2S, a member of the ubiquitin-conjugating enzyme (E2) family, exhibits oncogenic functions across multiple tumor types. By interacting with ubiquitin ligase TRIM21, UBE2S mediates K11-linked ubiquitination of LPP, thereby promoting lymph node metastasis in bladder cancer \u003csup\u003e[\u003cspan additionalcitationids=\"CR23 CR24 CR25 CR26\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e.Concurrently, studies have demonstrated that UBE2S promotes the progression of ovarian cancer. However, its precise functional roles and underlying mechanisms in colorectal cancer remain incompletely elucidated.\u003c/p\u003e\u003cp\u003eThe PI3K/AKT signaling pathway plays a pivotal regulatory role across diverse cell types. In malignant cells, this pathway is frequently aberrantly activated, thereby driving hyperproliferation through sustained oncogenic signaling \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e.Activation of PI3K phosphorylates AKT, triggering its translocation from the plasma membrane to the cytoplasm. This process subsequently activates downstream signaling cascades that promote cell survival and proliferation. The aberrant activation of the PI3K/AKT signaling pathway exerts profound effects on the malignant phenotypes of cancer cells. Emerging evidence has established that specific molecules drive tumorigenesis and progression through modulation of this pathway\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Notably, Xu et al. demonstrated that NETO2 regulates the PI3K/AKT axis to induce proliferation and metastatic dissemination in esophageal carcinoma cells\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e.Studies by Haines et al. have revealed that GREB1 modulates the PI3K/AKT signaling pathway to regulate proliferation in hormone-sensitive breast cancer\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn colorectal cancer, aberrant activation of the PI3K/AKT signaling pathway is closely associated with tumorigenesis, progression, and chemotherapy resistance. Inhibitors targeting this pathway have emerged as a promising therapeutic strategy \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Additionally, multiple genes have been demonstrated to influence colorectal cancer progression and treatment resistance through regulation of the PI3K/AKT signaling pathway. For instance, Claudin14 promotes colorectal cancer progression by activating the PI3K/AKT/mTOR pathway \u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Similarly, BEST4 exerts its oncogenic function in colorectal cancer via activation of the PI3K/Akt signaling pathway \u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Other molecules such as eIf3a, MUC3A, and LIN28B have also been confirmed to mediate malignant biological behaviors in colorectal cancer through modulation of the PI3K/AKT pathway \u003csup\u003e[\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Therefore, we conclude that activation of the PI3K/AKT signaling pathway is critically involved in the disease progression of colorectal cancer.\u003c/p\u003e\u003cp\u003eFirst, this study utilized GEO and TCGA datasets to conduct bioinformatics analysis on differentially expressed genes between normal colorectal tissues and colorectal cancer tissues. Further, LASSO and SVM-RFE methods were employed to identify characteristic genes of colorectal cancer, and UBE2S was found to be a characteristic gene of this malignancy. Through pan-cancer analysis of data from The Cancer Genome Atlas (TCGA) database, we observed that UBE2S is highly expressed in a variety of malignant tumors. In addition, the high expression of UBE2S showed a certain correlation with immune-infiltrating cells across pan-cancers. Therefore, we consider UBE2S to be a biologically meaningful gene that may play a role in tumor progression. Analysis of the GEPIA and TCGA databases revealed that UBE2S is differentially overexpressed in colorectal cancer and is associated with cellular immune infiltration. Subsequently, the expression of UBE2S in colorectal cancer was verified using Western blot. To clarify the significance of the differential expression of UBE2S, our experimental investigations demonstrated that knockdown of UBE2S inhibited the proliferation of colorectal cancer cells and increased their sensitivity to radiotherapy, whereas overexpression of UBE2S enhanced the proliferative capacity of cancer cells and reduced their sensitivity to radiotherapy. Furthermore, RNA sequencing analysis showed that UBE2S was significantly enriched in the PI3K/AKT pathway. We thus further explored the relationship between UBE2S and the PI3K/AKT signaling pathway, and the results indicated that UBE2S may promote the progression of colorectal cancer cells and their resistance to radiotherapy by activating the classical PI3K/AKT signaling pathway.\u003c/p\u003e\u003cp\u003eThis study confirms that UBE2S can enhance the proliferative capacity and radiotherapy resistance of colorectal cancer cells by activating the PI3K/AKT signaling pathway, though the specific mechanism underlying this process has not been explored in depth. Future research will focus on the specific manner in which UBE2S activates the PI3K/AKT signaling pathway. Based on the findings of this study, given that UBE2S functions as an oncogene in colorectal cancer, small-molecule inhibitors targeting UBE2S are expected to become a novel therapeutic option for patients with colorectal cancer.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn conclusion, we believe that UBE2S functions as an oncogene in colorectal cancer and can promote the progression and radiotherapy resistance of colorectal cancer by activating the PI3K/AKT pathway. Research on UBE2S not only helps to gain a deeper understanding of the molecular mechanisms underlying colorectal cancer but also provides potential targets and strategies for clinical treatment. Interventions targeting UBE2S may improve the prognosis and treatment outcomes of patients with colorectal cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYu Chang and Yufeng Hu designed and performed the experiments. Jing Guo and Yaqin Hao analyzed the experimental data. Fei Huang and Yu Liu wrote the paper. Yu Chang and Kai Bai revised the manuscript and submitted it for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Construction of Healthy Gastric Cancer Organs and Innovative Team of Clinical Application Research in Shaanxi Province[ 2025TD-20].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConfilict of interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article and its supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKlimeck L, Heisser T, Hoffmeister M, Brenner H. 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Oncogene. 2022;41(8):1166\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuo C, Wu D, Li X, Zhang Y, Hu B, Zhang T, Ren J, Wang T, Liu Y. eIf3a mediates malignant biological behaviors in colorectal cancer through the PI3K/AKT signaling pathway. Cancer Biol Ther. 2024;25(1):2355703.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSu W, Feng B, Hu L, Guo X, Yu M. MUC3A promotes the progression of colorectal cancer through the PI3K/Akt/mTOR pathway. BMC Cancer. 2022;22(1):602.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShin AE, Sugiura K, Kariuki SW, Cohen DA, Flashner SP, Klein-Szanto AJ, Nishiwaki N, De D, Vasan N, Gabre JT, Lengner CJ, Sims PA, Rustgi AK. LIN28B-mediated PI3K/AKT pathway activation promotes metastasis in colorectal cancer models. J Clin Invest. 2025;135(8):e186035.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"UBE2S, colorectal cancer, PI3K/AKT, proliferation, Radiosensitivity","lastPublishedDoi":"10.21203/rs.3.rs-7724776/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7724776/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eColorectal cancer is the third most common malignant tumor worldwide, accounting for approximately 10% of all newly diagnosed cancer cases annually. UBE2S has been demonstrated to play an oncogenic role in various types of cancer. however, its specific mechanism in colorectal cancer remains poorly understood.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eIn this study, we initially identified the differential expression of UBE2S between colorectal cancer tissues and normal colorectal tissues from patients by analyzing data from TCGA and GEO databases. Meanwhile, UBE2S also exhibits certain significance in pan-cancer. Further studies showed that compared with normal colorectal epithelial cells, UBE2S was highly expressed in colorectal cancer cells and had a certain correlation with immune infiltrating cells. We then investigated the effects of UBE2S knockdown and overexpression on the proliferation and radiotherapy sensitivity of colorectal cancer cells. RNA sequencing analysis revealed that UBE2S was enriched in pathways related to cell cycle, apoptosis, PI3K/AKT signaling, DNA replication, base excision repair, ferroptosis, and non-homologous end joining. Subsequently, we evaluated the changes in PI3K/AKT pathway-related proteins after UBE2S knockout and overexpression, as well as the effect of the PI3K/AKT pathway inhibitor LY294002 on the proliferation of colorectal cancer cells.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe results showed that UBE2S is highly expressed in colorectal cancer and has a certain correlation with immune infiltrating cells. UBE2S promotes the proliferation and radiotherapy resistance of colorectal cancer cells; meanwhile, overexpression of UBE2S can activate the PI3K/AKT pathway, and this effect can be reversed by the addition of LY294002. In conclusion, our findings suggest that UBE2S may promote the proliferation and radiotherapy resistance of colorectal cancer cells by activating the PI3K/AKT signaling pathway.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eIn this study, we believe that UBE2S acts as an oncogene in colorectal cancer and can promote the progression of colorectal cancer by activating the PI3K/AKT pathway. The study of UBE2S not only contributes to a deeper understanding of the molecular mechanisms of colorectal cancer but also provides potential targets and strategies for clinical treatment. Intervention targeting UBE2S may improve the prognosis and treatment outcomes of colorectal cancer patients.\u003c/p\u003e","manuscriptTitle":"From Data Mining to Functional Validation: UBE2S Regulates Proliferation and Radiosensitivity in Colorectal Cancer via the PI3K/AKT Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-29 18:37:15","doi":"10.21203/rs.3.rs-7724776/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":"21cc932b-ec8c-4543-ad0f-2963aa0315b8","owner":[],"postedDate":"October 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-27T00:09:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-29 18:37:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7724776","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7724776","identity":"rs-7724776","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}