The synergistic effect of TGM2 and TGFβ2 on the prognosis of colon cancer patients

The synergistic effect of TGM2 and TGFβ2 on the prognosis of colon cancer patients | 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 The synergistic effect of TGM2 and TGFβ2 on the prognosis of colon cancer patients Lei Shi, Yue Wang¹, Zhihua Cheng¹, Zhe Lv¹, Ri-feng Lu, Hai-cheng Gao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2781254/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 Colon cancer (CNC) ranks third in incidence and second in mortality among all cancers worldwide. Unfortunately, the available treatment options for CNC have limited effectiveness in improving patients' prognosis, and the pathogenesis of CNC remains poorly understood. Transglutaminase 2 (TGM2) and TGF-β2 both play a positive role in regulating cancer progression, promoting tumor progression and metastasis. However, the mutual relationship between TGM2 and TGF-β2 has not been well studied. Our analysis of transcriptome data from CNC patients on the TCGA website showed that the expression levels of TGF-β2 and TGM2 were related to TNM staging and tumor size (P < 0.05), and high expression of TGF-β2 and TGM2 was associated with a worse prognosis (P < 0.05). To validate these findings, we performed experiments using collected tumor tissue specimens from CNC patients, which demonstrated that inhibiting TGM2 could reduce the expression of TGF-β2 and improve the prognosis of CNC patients. Therefore, targeting TGM2 and TGF-β2 may become feasible therapeutic targets for CNC, and our study has provided directions for the development of CNC treatment drugs. colon cancer，TGM2，TGF-β2，immunohistochemistry Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 INTRODUCTION Colon cancer (CNC) is a common malignancy of the digestive system, ranking third in incidence worldwide after lung and breast cancers ( 1 ). Due to its lack of specific symptoms, CNC is often diagnosed at an advanced stage with metastasis, resulting in poor treatment outcomes ( 2 ). The median survival time for non-metastatic CNC patients is less than 3 years ( 3 ), and those with distant metastasis have even worse prognoses. Therefore, discovering new therapeutic targets is crucial for treating CNC. Transglutaminase 2 (TGM2) is a GTP-binding enzyme with acyltransferase activity ( 9 ). It can shuttle between the cytoplasm and nucleus to assist in cell signaling and participate in important cellular activities such as cell adhesion, epithelial-mesenchymal transition (EMT), cell growth, and migration ( 10 , 12 ). TGM2 can promote tumor metastasis by inducing EMT ( 4 – 5 ). In the normal human body, the TGF-β (transforming growth factor β) signaling pathway regulates cell growth, differentiation, and immune response ( 8 ). In cancer, TGF-β has a dual role, acting as a tumor suppressor in early stages and promoting tumor invasion and metastasis with high expression in late stages ( 8 ). Therefore, high expression of TGF-β is associated with poorer clinical prognosis. TGF-β has three subtypes, TGF-β1, TGF-β2, and TGF-β3 ( 7 ), among which TGF-β2 is involved in regulating cancer progression, inducing EMT, and leading to poor prognosis ( 15 ). However, the relationship between TGM2 and TGF-β2 has not been studied to date. In this study, we used bioinformatics methods to compare the expression of TGM2 and TGF-β2 in tumor tissue and adjacent normal tissue. We also conducted external verification experiments such as HE staining, immunohistochemistry, immunocytochemistry, and protein immunoblotting to further investigate the correlation between TGM2 and TGF-β2. Finally, we discussed the potential value of targeting TGM2 and TGF-β2 in the treatment of CNC. MATERIALS AND METHODS Data Acquisition CNC transcriptome and phenotype data were downloaded from the TCGA website ( https://portal.gdc.cancer.gov/ ). Clinical sample data for CNC were obtained from the UCSC Xena website ( https://xenabrowser.net/hub/ ), and tumor and normal samples were obtained after excluding samples lacking survival data. TCGA Ethics and Policy permits open access to TCGA data. Expression and Survival Analysis First, the transcriptome data in the TCGA database were analyzed. The expression levels of the TGM2 and TGF-β2 genes in tumor and normal tissues were compared using Welch's t-test (P < 0.01). Box plots were used to visualize the expression results in tumor and normal tissues. The chi-squared test was used to verify the correlation between the expression levels of TGF-β2 and TGM2 and TNM stage and maximum tumor diameter (P < 0.05). Kaplan-Meier curves were used to compare the overall survival (OS) and disease specific survival (DSS) of the high and low expression groups (P < 0.05). Tissue Specimen Acquisition From 2016 to 2020, we collected a total of 30 CNC tissue samples from the Sino-Japanese Union Hospital of Jilin University. All samples were pathologically diagnosed as colon cancer tissue. This study was approved by the Ethics Committee of the Sino-Japanese Union Hospital of Jilin University. We followed the Helsinki Declaration, and all patients provided written informed consent before specimen collection. Experimental reagents We used rabbit anti-human TGF-β2 polyclonal antibody, TGM2 antibody, and goat anti-rabbit polyclonal secondary antibody purchased from Protein Tech Group Inc. The immunohistochemistry kit was provided by Fuzhou Maixin Biotech Co., Ltd., and the anti-fluorescence quenching mounting solution was provided by Shanghai Biyun Tian Biotech Co., Ltd. HE(Hematoxylin and eosin)staining HE staining is a routine histological staining method used to display tissue structures and cell morphology. In this process, the pathological sections were soaked in 10% formalin for 30 minutes and embedded in paraffin. Then, they were washed with 95%, 80%, and 75% ethanol for 1 minute each. Finally, the samples were rinsed with tap water for 1 minute and images were collected. TGF-β2 immunohistochemistry staining Immunohistochemical staining is a technique that adds antibody labeling to specific proteins to observe their distribution and expression levels in pathological sections. In this process, paraffin-embedded sections are first treated with xylene and hydrated in a descending alcohol series. Then, 0.3% hydrogen peroxide formaldehyde solution was added to a pepsin-digested smear at 37˚C. After 15 minutes, the samples are sealed, washed and treated with PBS(Phosphate buffered saline) buffer and BSA (Bovine serum albumin). TGF-β2 polyclonal antibody is added to the sample in a 1:300 ratio, incubated at 4°C, and detected using a universal secondary antibody reagent and DAB (Horseradish peroxidase Color Developing kit) staining. Finally, photos are taken to observe the results. Cell culture Cell culture is the process of placing cells in suitable culture media to support their growth and reproduction. Sw480 cells are human colon cancer cells, provided by professor Chongyang Liang from Institute of Regenerative Medicine, Jilin University. SW480 cells are cultured in DMEM (Dulbecco's modified eagle medium) medium with the addition of 10% FBS (Fetal Bovine Serum), 100 U/mL penicillin G, and 100 µg/mL streptomycin, and cultured in a humidified incubator with 5% CO2 at 37℃. SW480 cell lines are randomly divided into an untreated group, high, medium, and low cisplatin groups, and a TGM2 inhibitor group for further experimental research. MTT assay (Cisplatin) The MTT assay can be used to detect cell survival and growth. SW480 cells were seeded in a 96-well plate and treated with 1 or 2mg/L of cisplatin for 24 hours. MTT solution was then added and incubated for 30 minutes before analyzing the cell survival rate at 450 nm. Immunofluorescence Immunofluorescence was used to detect the protein expression levels of TGM2 and TGF-β2 in SW480 cells after processed with cisplatin. The slices were subjected to double immunofluorescence staining under the same conditions. β-actin, TGM2 and TGF-β2 polyclonal antibodies (1:300) were used to stain fixed cell slices, followed by incubation with anti-rabbit FITC (Fluorescein isothiocyanate isomer) secondary antibody (1:1,000. Sigma-Aldrich. Merck KGaA). Western blot analysis SW480 cell lines and human tissue samples were lysed with cell lysis buffer containing 1 ml of phenylmethylsulfonyl fluoride. After centrifugation at 12,000 rpm for 15 min, the supernatant was mixed with 5X buffer (SDS-PAGE protein loading buffer) and heated for 10 min. Then, 30µg of total protein was separated using 10% SDS-PAGE protein loading buffer. After blocking with 5% skim milk membrane for 1 hour, the protein was incubated with β-actin, TGM2, and TGF-β2 polyclonal antibodies. Statistical analysis Continuous variables were expressed as mean ± standard deviation. Statistical analysis was performed using SPSS software, version 19.0. Group comparisons were performed using t-test, while one-way analysis of variance (ANOVA) was used for intergroup comparisons between all treatment groups and the untreated control group, followed by Dunnett's post hoc test. Differences were considered statistically significant at P < 0.05. RESULTS Expression analysis of TGM2 and TGF-β2 As shown in Fig. 1 A-B, the expression levels of TGF-β2 and TGM2 in tumor tissues were significantly higher than those in normal tissues (Welch's t-test, P < 0.01). In addition, as shown in Fig. 1 C-D, the expression levels of TGF-β2 and TGM2 were correlated with TNM stage and tumor maximum diameter (P < 0.05). As shown in Table 1, there was no significant difference in clinical characteristics between patients with high and low expression of TGF-β2. Table 1. Chi-square test for clinical characteristics between high and low TGF-β2 expression groups. TGF-β 2 P-value Low Expression High Expression Age(y) ≥ 60 29 37 0.184 < 60 66 56 gender Female 32 52 0.002 male 63 41 pathologic T T1 5 2 0.043 T2 20 9 T3 60 65 T4 9 16 pathologic N N0 64 41 0.007 N1 18 25 N2 13 25 pathologic M M0 64 62 0.099 M1 9 18 lymphatic invasion No 58 45 0.088 Yes 25 34 longest dimension(cm) > 1 18 9 0.009 ≤ 1 17 31 Statistical analysis was based on chi-square test (P < = 0.01). Kaplan-Meier survival analysis As shown in Fig. 2 , in the survival analysis of TGF-β2 and TGM2, patients with high expression had lower overall survival (OS) and disease specific survival (DSS) than those with low expression. The results above indicate that high expression of TGF-β2 and TGM2 has a negative impact on patient prognosis. HE staining Tissue biopsy is the gold standard for diagnosing tumors, and the most commonly used method is HE staining. The 30 tissue samples we collected were confirmed to be colon cancer tissue by HE staining. HE staining results showed that the cells in adjacent normal tissues were neatly arranged, without swelling and with clear structures (Fig. 3 A), while the tumor tissues showed glandular-like arrangement, with moderate dysplasia and stromal fibrosis, and obvious changes in histological morphology (Fig. 3 B). Immunohistochemistry staining Immunohistochemistry staining is used to determine the location and relative content of antigens in tissues or cells. The expression level of TGF-β2 in CNCtissues was higher than that in adjacent normal tissues (Fig. 4 C). TGF-β2 in adjacent normal tissues showed negative/weak positive expression (Fig. 4 A). Positive expression of TGF-β2 was brown or yellow, mainly concentrated in the nucleus, cytoplasm, and a small amount in the cell membrane (Fig. 4 B). The expression level of TGM2 in colon cancer tissues was higher than that in adjacent normal tissues. Western blot analysis The expression level of TGF-β2 in tumor tissue was significantly higher than that in adjacent normal tissue (P < 0.05) (Fig. 5 ). MTT assay (cisplatin) We treated SW480 cells with different concentrations of cisplatin to detect cell viability. The results showed that the survival rate of SW480 cells treated with cisplatin for 24 hours was decreased compared to untreated SW480 cells (P < 0.05) (Fig. 6 ). Effect of cisplatin on TGF-β2 expression in SW480 cells As shown in the figure, the fluorescence intensity of TGF-β2 in SW480 cells treated with 1 mg/L and 2 mg/L cisplatin was decreased compared to untreated SW480 cells, mainly concentrated in the nucleus and cytoplasm (P < 0.05) (Figs. 7 A-F). The cell structure also underwent significant changes after cisplatin treatment. DAPI (4,6-Diamidino-2-phenylindole), a fluorescent dye that can strongly bind to DNA, was used. Expression levels of TGM2 and TGF-β2 after treatment with TGM2 inhibitor The fluorescence intensity of TGM2 in SW480 cells without TGM2 inhibitor treatment was stronger than that in cells treated with TGM2 inhibitor (P < 0.05) (Fig. 8 A-D). The TGM2 inhibitor used was MDC (Dansylcadaverine) at a concentration of 150 µmol/L. After treatment with the TGM2 inhibitor, the fluorescence intensity of TGF-β2 in SW480 cells was weaker than that in untreated cells (P < 0.05) (Fig. 9 A-D). These results indicate a close correlation between the expression levels of TGM2 and TGF-β2. DISCUSSION After analyzing TCGA data, we discovered that the gene expression levels of TGM2 and TGF-β2 were significantly higher in CNC tumor tissue than in normal tissue. Additionally, high expression of TGM2 and TGF-β2 was linked to a worse prognosis, as evidenced by lower overall survival (OS) time and shorter disease specific survival time (DSS) based on Kaplan-Meier survival analysis. This association was likely due to the correlation between the expression levels of TGF-β2 and TGM2 with tumor TNM stage and maximum diameter (P < 0.05), which ultimately led to a worse prognosis for CNC patients. TGM2 is a multifunctional protein that participates in numerous cellular processes, such as apoptosis, inflammation, cell proliferation, and signal transduction ( 4 ). In terms of cancer development, high expression of TGM2 is linked to cancer progression and resistance ( 4 , 14 ). TGM2 and TGF-β2 both promote epithelial-mesenchymal transition (EMT) of tumor cells, which is a driving force of tumor metastasis and is linked to tumor cell invasiveness ( 8 , 14 ). Furthermore, TGM2 can activate NF-κB and TGF-β2 can induce lipid droplet formation to induce EMT ( 4 , 8 , 14 , 15 ). KANG et al. discovered that high expression of TGM2 promoted infiltration and metastasis of CNC, and knocking out TGM2 inhibited tumor cell growth ( 5 ). Therefore, the regulatory role of TGM2 is crucial for CNC, and given that the expression level of TGM2 in tumor tissue is much higher than in normal tissue, it is a promising therapeutic target for CNC. TGF-β2 belongs to the TGF-β superfamily and participates in the regulation of human metabolism, cell growth, and disease development ( 8 , 16 – 18 ). TGF-β2 also promotes cancer progression ( 15 ) and is associated with poor prognosis in patients. The TGF-β superfamily has a positive effect on TGM2 expression ( 4 ), and TGM2 and TGF-β2 also have a close mutual regulatory relationship. Our experimental data confirmed the correlation between TGM2 and TGF-β2, and we found that inhibiting TGM2 expression resulted in decreased expression of TGF-β2. We believe that inhibiting the expression of TGM2 may inhibit the expression of TGF-β2, thereby further affecting the occurrence and development of CNC. Additionally, cisplatin was found to inhibit the expression of TGF-β2, indicating that inhibiting TGM2 could be an effective treatment method for CNC. Preliminary results have shown promise in anti-tumor therapy targeting TGM2 ( 13 ). Our analysis of TCGA data revealed a significant upregulation of TGM2 and TGF-β2 expression levels in CNC tumor tissue compared to normal tissue. Moreover, patients with high expression of TGM2 and TGF-β2 exhibited worse prognosis, indicating their involvement in the progression of CNC. Our results also suggest that TGM2 and TGF-β2 expression levels were correlated with TNM stage and maximum diameter of the tumor (P < 0.05), which could affect the prognosis of the patient. TGM2 and TGF-β2 were found to promote tumor metastasis by activating EMT, making TGM2 an ideal therapeutic target for CNC. These findings have important clinical significance for the treatment. However, our study has some limitations, including the absence of further clinical trials and insufficient sample size to account for accidental errors. We plan to address these limitations in future studies. Overall, our findings have important clinical implications for the treatment of CNC. Declarations Acknowledgements Thanks to all the patients involved in this study, department of Clinical Pharmacy, School of Pharmaceutical Sciences, Jilin University, department of Gastroenterology, the First Hospital of Jilin University, department of Thoracic surgery, China-Japan Union Hospital of Jilin University. Authors’ Contributions Hai-cheng Gao, Yue Wang and Lei Shi contributed to the conception of the study, Zhe Lv and performed the Bioinformatics analysis and experiment, Lei Shi and Yue Wang contributed significantly to analysis and manuscript preparation, Zhi Hua Cheng performed the data analyses, Lei Shi wrote the manuscript, RiFeng Lu helped perform the analysis with constructive discussions. Funding This work was supported by Jilin University Fund Support (grant number 4150102000049,415010300074), Changchun Science and Technology Bureau (19SS015), the project comes from the Department of science and technology of Jilin Province (grant number 20200201459jc), Jilin Province direct health special project (grant number JLSWSRCZX2020-008), Jilin Provincial Finance Department (grant number JLSWSRCZX2020-083), Education Department of Jilin Province (grant number JJKH20211209KJ). Availability of data and materials The data used and/or analysed in this study were downloaded from the Cancer Genome Atlas public database, available at: https://portal.gdc.cancer.gov/. Ethics approval and consent to participate This trial was conducted in accordance with the principles outlined in the World Medical Association Declaration of Helsinki for experiments involving human subjects, as revised in 2013. This study was approved by the Research Ethics Committee of China-Japan Union Hospital of Jilin University. Patients gave written informed consent before specimen collection, and the experimental data and personal information they provided were kept completely confidential. Consent for publication Not applicable. Competing interests The authors declare that there is no economic and non-economic competitive interest associated with research. Author details ¹ (Department of Clinical Pharmacy, School of Pharmaceutical Sciences, Jilin University, Changchun. Department of Gastroenterology, the First Hospital of Jilin University, Changchun, China. Department of Thoracic surgery, China-Japan Union Hospital of Jilin University, Changchun, China.) References Baidoun F, Elshiwy K, Elkeraie Y, Merjaneh Z, Khoudari G, Sarmini MT, Gad M, Al-Husseini M, Saad A. Colorectal Cancer Epidemiology: Recent Trends and Impact on Outcomes. Curr Drug Targets. 2021;22(9):998-1009. doi: 10.2174/1389450121999201117115717. PMID: 33208072. Li H, Li L, Huang X, Li Y, Zou T, Zhuo X, Chen Y, Liu Y, Tang Y. Radiotherapy-induced dysphagia and its impact on quality of life in patients with nasopharyngeal carcinoma. Strahlenther Onkol. 2019 Jun;195(6):457-467. English. doi: 10.1007/s00066-018-01421-6. Epub 2019 Jan 28. PMID: 30689027. Dekker E, Tanis PJ, Vleugels JLA, Kasi PM, Wallace MB. Colorectal cancer. Lancet. 2019 Oct 19;394(10207):1467-1480. doi: 10.1016/S0140-6736(19)32319-0. PMID: 31631858. Eckert RL, Kaartinen MT, Nurminskaya M, Belkin AM, Colak G, Johnson GV, Mehta K. Transglutaminase regulation of cell function. Physiol Rev. 2014 Apr;94(2):383-417. doi: 10.1152/physrev.00019.2013. PMID: 24692352; PMCID: PMC4044299. Kang S, Oh SC, Min BW, Lee DH. Transglutaminase 2 Regulates Self-renewal and Stem Cell Marker of Human Colorectal Cancer Stem Cells. Anticancer Res. 2018 Feb;38(2):787-794. doi: 10.21873/anticanres.12285. PMID: 29374703. Shan H, Zhou X, Chen C. MicroRNA‑214 suppresses the viability, migration and invasion of human colorectal carcinoma cells via targeting transglutaminase 2. Mol Med Rep. 2019 Aug;20(2):1459-1467. doi: 10.3892/mmr.2019.10325. Epub 2019 Jun 3. PMID: 31173203; PMCID: PMC6625444. Morikawa M, Derynck R, Miyazono K. TGF-β and the TGF-β Family: Context-Dependent Roles in Cell and Tissue Physiology. Cold Spring Harb Perspect Biol. 2016 May 2;8(5):a021873. doi: 10.1101/cshperspect.a021873. PMID: 27141051; PMCID: PMC4852809. Syed V. TGF-β Signaling in Cancer. J Cell Biochem. 2016 Jun;117(6):1279-87. doi: 10.1002/jcb.25496. Epub 2016 Feb 11. PMID: 26774024. Bae GH, Kim YS, Park JY, Lee M, Lee SK, Kim JC, Kim JG, Shin YJ, Lee H, Kim SY, Bae YS, Zabel BA, Kim HS, Bae YS. Unique characteristics of lung-resident neutrophils are maintained by PGE2/PKA/Tgm2-mediated signaling. Blood. 2022 Aug 25;140(8):889-899. doi: 10.1182/blood.2021014283. PMID: 35679477; PMCID: PMC9412003. Maggio N, Sellitti S, Capano CP, Papa M. Tissue-transglutaminase in rat and human brain: light and electron immunocytochemical analysis and in situ hybridization study. Brain Res Bull. 2001 Oct-Nov 1;56(3-4):173-82. doi: 10.1016/s0361-9230(01)00649-9. PMID: 11719248. Liu H, Jiang B. Let-7a-5p represses proliferation, migration, invasion and epithelial-mesenchymal transition by targeting Smad2 in TGF-b2-induced human lens epithelial cells. J Biosci. 2020;45:59. PMID: 32345785. Shrestha R, Tatsukawa H, Shrestha R, Ishibashi N, Matsuura T, Kagechika H, Kose S, Hitomi K, Imamoto N, Kojima S. Molecular mechanism by which acyclic retinoid induces nuclear localization of transglutaminase 2 in human hepatocellular carcinoma cells. Cell Death Dis. 2015 Dec 3;6(12):e2002. doi: 10.1038/cddis.2015.339. PMID: 26633708; PMCID: PMC4720877. Kang JH, Lee SH, Kim SY. Discovery of a novel target for renal cell carcinoma: transglutaminase 2. Cell Death Dis. 2016 Apr 21;7(4):e2200. doi: 10.1038/cddis.2016.99. PMID: 27100894; PMCID: PMC4855678. Das V, Bhattacharya S, Chikkaputtaiah C, Hazra S, Pal M. The basics of epithelial-mesenchymal transition (EMT): A study from a structure, dynamics, and functional perspective. J Cell Physiol. 2019 Sep;234(9):14535-14555. doi: 10.1002/jcp.28160. Epub 2019 Feb 5. PMID: 30723913. Corbet C, Bastien E, Santiago de Jesus JP, Dierge E, Martherus R, Vander Linden C, Doix B, Degavre C, Guilbaud C, Petit L, Michiels C, Dessy C, Larondelle Y, Feron O. TGFβ2-induced formation of lipid droplets supports acidosis-driven EMT and the metastatic spreading of cancer cells. Nat Commun. 2020 Jan 23;11(1):454. doi: 10.1038/s41467-019-14262-3. PMID: 31974393; PMCID: PMC6978517. Takahashi H, Alves CRR, Stanford KI, Middelbeek RJW, Nigro P, Ryan RE, Xue R, Sakaguchi M, Lynes MD, So K, Mul JD, Lee MY, Balan E, Pan H, Dreyfuss JM, Hirshman MF, Azhar M, Hannukainen JC, Nuutila P, Kalliokoski KK, Nielsen S, Pedersen BK, Kahn CR, Tseng YH, Goodyear LJ. TGF-β2 is an exercise-induced adipokine that regulates glucose and fatty acid metabolism. Nat Metab. 2019 Feb;1(2):291-303. doi: 10.1038/s42255-018-0030-7. Epub 2019 Feb 11. PMID: 31032475; PMCID: PMC6481955. Dropmann A, Dooley S, Dewidar B, Hammad S, Dediulia T, Werle J, Hartwig V, Ghafoory S, Woelfl S, Korhonen H, Janicot M, Wosikowski K, Itzel T, Teufel A, Schuppan D, Stojanovic A, Cerwenka A, Nittka S, Piiper A, Gaiser T, Beraza N, Milkiewicz M, Milkiewicz P, Brain JG, Jones DEJ, Weiss TS, Zanger UM, Ebert M, Meindl-Beinker NM. TGF-β2 silencing to target biliary-derived liver diseases. Gut. 2020 Sep;69(9):1677-1690. doi: 10.1136/gutjnl-2019-319091. Epub 2020 Jan 28. PMID: 31992593; PMCID: PMC7456737. Sun Y, Xiong L, Wang X, Wang L, Chen B, Huang J, Huang M, Chen J, Wu J, Huang S, Liu Y. Autophagy inhibition attenuates TGF-β2-induced epithelial-mesenchymal transition in lens epithelial cells. Life Sci. 2021 Jan 15;265:118741. doi: 10.1016/j.lfs.2020.118741. Epub 2020 Nov 10. PMID: 33181173. Additional Declarations No competing interests reported. Supplementary Files TGF2.docx TGM2.docx 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-2781254","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":197848943,"identity":"bfccd3aa-c4fe-400d-97f3-e88ce46c8e8f","order_by":0,"name":"Lei Shi","email":"","orcid":"","institution":"Department of Gastroenterology, the First Hospital of Jilin University, Changchun","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Shi","suffix":""},{"id":197848945,"identity":"b673e1bb-8e06-4059-8059-1670924acf92","order_by":1,"name":"Yue Wang¹","email":"","orcid":"","institution":"Department of Thoracic surgery, China-Japan Union Hospital of Jilin University, Changchun","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Wang¹","suffix":""},{"id":197848948,"identity":"3127d2f9-6657-4072-9ef1-591542987ee5","order_by":2,"name":"Zhihua Cheng¹","email":"","orcid":"","institution":"Vascular Surgery Department, General Surgery Center, First Hospital of Jilin University, ChangChun","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhihua","middleName":"","lastName":"Cheng¹","suffix":""},{"id":197848950,"identity":"5ae8df1c-113d-4fd2-8478-b73a08f2d407","order_by":3,"name":"Zhe Lv¹","email":"","orcid":"","institution":"Department of Clinical Pharmacy, School of Pharmaceutical Sciences, Jilin University, Changchun","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhe","middleName":"","lastName":"Lv¹","suffix":""},{"id":197848952,"identity":"5b14871d-1587-4ce8-a0f6-ae496e987dff","order_by":4,"name":"Ri-feng Lu","email":"","orcid":"","institution":"School of Public Health, the First Hospital of Jilin University, Changchun","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ri-feng","middleName":"","lastName":"Lu","suffix":""},{"id":197848954,"identity":"7f4ff261-0f85-4230-b831-de845efaccd5","order_by":5,"name":"Hai-cheng Gao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIie3Pv0oDQRDH8TkWxubQdo7A7StMWPAPprPwNXYRLk0KyxSiK4GrAvcCkmdIZb2wsDYHtpY5BSsLK7nCQpNWuE1psd9ymQ8/FiCV+p8hAE9KBP/U6zmVR/uR60odZkHTpj1Vhd2LfHqzEi0XXT036xiRlz5sevYK0TGbFWVrEN3rywAZt9V0vORpibnT2jySOAFUajZE7Ox4BHyukJxzvwTPbI6jQdJ8bIkwtezurXmgnF2ESNqtXJgavABjiaKE6b0qllwphICgA3GxiPxFNleB+u9JKe3zV9bf3N41B4vubXDF/XkSA+e7FRs5SKVSqRT8AKYNSxW9+V1uAAAAAElFTkSuQmCC","orcid":"","institution":"Department of Clinical Pharmacy, School of Pharmaceutical Sciences, Jilin University, Changchun","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Hai-cheng","middleName":"","lastName":"Gao","suffix":""}],"badges":[],"createdAt":"2023-04-05 13:29:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2781254/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2781254/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":36748427,"identity":"23a4514e-8316-4386-8e91-8feca0855fd6","added_by":"auto","created_at":"2023-05-09 16:09:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1334163,"visible":true,"origin":"","legend":"\u003cp\u003eA. Gene expression analysis of TGF-β2, B. TGM2 in normal and tumor tissues, C. GTEX (Genotype-Tissue Expression) of TGF-β2 in colon cancer tissue，D. GTEX of TGM2 in colon cancer tissue.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2781254/v1/85b095df2ba5a0ee971f185a.png"},{"id":36748430,"identity":"880cff71-5c1c-4803-a5d3-6f1861cec86a","added_by":"auto","created_at":"2023-05-09 16:09:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":947478,"visible":true,"origin":"","legend":"\u003cp\u003eA. Kaplan-Meier curves of overall survival and disease-specific survival for TGF-β2, B. Kaplan-Meier curves of overall survival and disease-specific survival for TGM2.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2781254/v1/871ce43b5fb15dff7a0b1f4d.png"},{"id":36748877,"identity":"178436db-fa87-42cd-a162-a696a0cef645","added_by":"auto","created_at":"2023-05-09 16:17:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1925185,"visible":true,"origin":"","legend":"\u003cp\u003eHE staining. A. Paracancerous tissue, B. Colon cancer tissue.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2781254/v1/bdb9db3e47fd16ecb0502bab.png"},{"id":36748876,"identity":"4aa13a66-dd58-4837-8964-0341ec363f50","added_by":"auto","created_at":"2023-05-09 16:17:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2128708,"visible":true,"origin":"","legend":"\u003cp\u003eA. Expression of TGF-β2 in adjacent normal tissue (magnification 200×), B. Expression of TGF-β2 in CNC tissue (magnification 200×), C. Histogram of TGF-β2 expression levels in CNC tissue and adjacent normal tissue (P\u0026lt;0.01).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2781254/v1/389aca23ca7a589ac37f5ab1.png"},{"id":36748878,"identity":"f713e1f5-3721-4349-9faf-b2573650cdaf","added_by":"auto","created_at":"2023-05-09 16:17:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":250656,"visible":true,"origin":"","legend":"\u003cp\u003eProtein blot analysis of TGF-β2 expression in colon cancer tissue and adjacent tissue.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-2781254/v1/cdfe4ed06b98f14466f42260.png"},{"id":36749263,"identity":"0b84698e-db88-466a-b9b1-537fd615db85","added_by":"auto","created_at":"2023-05-09 16:25:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":121885,"visible":true,"origin":"","legend":"\u003cp\u003eViability of SW480 cells treated with cisplatin.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-2781254/v1/b458d33ca159fa5bb184e2ac.png"},{"id":36748435,"identity":"122105a7-7d7d-4b66-845a-abc27a94e313","added_by":"auto","created_at":"2023-05-09 16:09:32","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":568590,"visible":true,"origin":"","legend":"\u003cp\u003eA. DAPI expression in untreated SW480 cells, B. DAPI expression in SW480 cells treated with 1mg/L cisplatin, C. DAPI expression in SW480 cells treated with 2mg/L cisplatin, D. DAPI expression in untreated SW480 cells, E. TGF-β2 expression in SW480 cells treated with 1mg/L cisplatin, F. TGF-β2 expression in SW480 cells treated with 2mg/L cisplatin.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-2781254/v1/2caf21cfbdd31674ded998b5.png"},{"id":36748879,"identity":"44fe9239-a008-4ce6-b540-29c43aa3b5d6","added_by":"auto","created_at":"2023-05-09 16:17:32","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":566936,"visible":true,"origin":"","legend":"\u003cp\u003eA. Expression of DAPI in SW480 cells without TGM2 inhibitor treatment, B. Expression of DAPI in SW480 cells treated with TGM2 inhibitor, C. Expression of TGM2 in SW480 cells without TGM2 inhibitor treatment, D. Expression of TGM2 in SW480 cells treated with TGM2 inhibitor.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-2781254/v1/d53db44da166bfc1da498c66.png"},{"id":36748433,"identity":"4695c608-7b5d-456b-ae8f-9b0639bcd576","added_by":"auto","created_at":"2023-05-09 16:09:32","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1291526,"visible":true,"origin":"","legend":"\u003cp\u003eA. Expression of DAPI in untreated SW480 cells without TGM2 inhibitor treatment. B. Expression of DAPI in SW480 cells treated with TGM2 inhibitor. C. Expression of TGF-β2 in SW480 cells without TGM2 inhibitor treatment, D. Expression of TGF-β2 in SW480 cells treated with TGM2 inhibitor.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-2781254/v1/960eb33a1be2a0b038ba2141.png"},{"id":49007994,"identity":"642cde56-f851-44c7-98fc-f4a5cec15117","added_by":"auto","created_at":"2023-12-31 08:07:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4974523,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2781254/v1/fcd10186-2b86-4238-a3ec-211499302a26.pdf"},{"id":36748874,"identity":"305220b9-7e9a-4d79-8208-e0625f4a751f","added_by":"auto","created_at":"2023-05-09 16:17:32","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":54008,"visible":true,"origin":"","legend":"","description":"","filename":"TGF2.docx","url":"https://assets-eu.researchsquare.com/files/rs-2781254/v1/89332c032fbaed8e5cb70ea6.docx"},{"id":36748875,"identity":"1ece255d-d23e-4386-917e-a034e8c065a5","added_by":"auto","created_at":"2023-05-09 16:17:32","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":33076,"visible":true,"origin":"","legend":"","description":"","filename":"TGM2.docx","url":"https://assets-eu.researchsquare.com/files/rs-2781254/v1/a80b41763831870d7d3ab317.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The synergistic effect of TGM2 and TGFβ2 on the prognosis of colon cancer patients","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eColon cancer (CNC) is a common malignancy of the digestive system, ranking third in incidence worldwide after lung and breast cancers (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Due to its lack of specific symptoms, CNC is often diagnosed at an advanced stage with metastasis, resulting in poor treatment outcomes (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The median survival time for non-metastatic CNC patients is less than 3 years (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), and those with distant metastasis have even worse prognoses. Therefore, discovering new therapeutic targets is crucial for treating CNC.\u003c/p\u003e \u003cp\u003eTransglutaminase 2 (TGM2) is a GTP-binding enzyme with acyltransferase activity (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). It can shuttle between the cytoplasm and nucleus to assist in cell signaling and participate in important cellular activities such as cell adhesion, epithelial-mesenchymal transition (EMT), cell growth, and migration (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). TGM2 can promote tumor metastasis by inducing EMT (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). In the normal human body, the TGF-β (transforming growth factor β) signaling pathway regulates cell growth, differentiation, and immune response (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). In cancer, TGF-β has a dual role, acting as a tumor suppressor in early stages and promoting tumor invasion and metastasis with high expression in late stages (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Therefore, high expression of TGF-β is associated with poorer clinical prognosis. TGF-β has three subtypes, TGF-β1, TGF-β2, and TGF-β3 (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), among which TGF-β2 is involved in regulating cancer progression, inducing EMT, and leading to poor prognosis (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). However, the relationship between TGM2 and TGF-β2 has not been studied to date.\u003c/p\u003e \u003cp\u003eIn this study, we used bioinformatics methods to compare the expression of TGM2 and TGF-β2 in tumor tissue and adjacent normal tissue. We also conducted external verification experiments such as HE staining, immunohistochemistry, immunocytochemistry, and protein immunoblotting to further investigate the correlation between TGM2 and TGF-β2. Finally, we discussed the potential value of targeting TGM2 and TGF-β2 in the treatment of CNC.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eData Acquisition\u003c/h2\u003e \u003cp\u003eCNC transcriptome and phenotype data were downloaded from the TCGA website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://portal.gdc.cancer.gov/\u003c/span\u003e\u003cspan address=\"https://portal.gdc.cancer.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Clinical sample data for CNC were obtained from the UCSC Xena website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://xenabrowser.net/hub/\u003c/span\u003e\u003cspan address=\"https://xenabrowser.net/hub/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and tumor and normal samples were obtained after excluding samples lacking survival data. TCGA Ethics and Policy permits open access to TCGA data.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eExpression and Survival Analysis\u003c/h2\u003e \u003cp\u003eFirst, the transcriptome data in the TCGA database were analyzed. The expression levels of the TGM2 and TGF-β2 genes in tumor and normal tissues were compared using Welch's t-test (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Box plots were used to visualize the expression results in tumor and normal tissues. The chi-squared test was used to verify the correlation between the expression levels of TGF-β2 and TGM2 and TNM stage and maximum tumor diameter (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Kaplan-Meier curves were used to compare the overall survival (OS) and disease specific survival (DSS) of the high and low expression groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTissue Specimen Acquisition\u003c/h2\u003e \u003cp\u003eFrom 2016 to 2020, we collected a total of 30 CNC tissue samples from the Sino-Japanese Union Hospital of Jilin University. All samples were pathologically diagnosed as colon cancer tissue. This study was approved by the Ethics Committee of the Sino-Japanese Union Hospital of Jilin University. We followed the Helsinki Declaration, and all patients provided written informed consent before specimen collection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eExperimental reagents\u003c/h2\u003e \u003cp\u003eWe used rabbit anti-human TGF-β2 polyclonal antibody, TGM2 antibody, and goat anti-rabbit polyclonal secondary antibody purchased from Protein Tech Group Inc. The immunohistochemistry kit was provided by Fuzhou Maixin Biotech Co., Ltd., and the anti-fluorescence quenching mounting solution was provided by Shanghai Biyun Tian Biotech Co., Ltd.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eHE(Hematoxylin and eosin)staining\u003c/h2\u003e \u003cp\u003eHE staining is a routine histological staining method used to display tissue structures and cell morphology. In this process, the pathological sections were soaked in 10% formalin for 30 minutes and embedded in paraffin. Then, they were washed with 95%, 80%, and 75% ethanol for 1 minute each. Finally, the samples were rinsed with tap water for 1 minute and images were collected.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTGF-β2 immunohistochemistry staining\u003c/h2\u003e \u003cp\u003eImmunohistochemical staining is a technique that adds antibody labeling to specific proteins to observe their distribution and expression levels in pathological sections. In this process, paraffin-embedded sections are first treated with xylene and hydrated in a descending alcohol series. Then, 0.3% hydrogen peroxide formaldehyde solution was added to a pepsin-digested smear at 37˚C. After 15 minutes, the samples are sealed, washed and treated with PBS(Phosphate buffered saline) buffer and BSA (Bovine serum albumin). TGF-β2 polyclonal antibody is added to the sample in a 1:300 ratio, incubated at 4\u0026deg;C, and detected using a universal secondary antibody reagent and DAB (Horseradish peroxidase Color Developing kit) staining. Finally, photos are taken to observe the results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eCell culture is the process of placing cells in suitable culture media to support their growth and reproduction. Sw480 cells are human colon cancer cells, provided by professor Chongyang Liang from Institute of Regenerative Medicine, Jilin University. SW480 cells are cultured in DMEM (Dulbecco's modified eagle medium) medium with the addition of 10% FBS (Fetal Bovine Serum), 100 U/mL penicillin G, and 100 \u0026micro;g/mL streptomycin, and cultured in a humidified incubator with 5% CO2 at 37℃. SW480 cell lines are randomly divided into an untreated group, high, medium, and low cisplatin groups, and a TGM2 inhibitor group for further experimental research.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMTT assay (Cisplatin)\u003c/h2\u003e \u003cp\u003eThe MTT assay can be used to detect cell survival and growth. SW480 cells were seeded in a 96-well plate and treated with 1 or 2mg/L of cisplatin for 24 hours. MTT solution was then added and incubated for 30 minutes before analyzing the cell survival rate at 450 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eImmunofluorescence was used to detect the protein expression levels of TGM2 and TGF-β2 in SW480 cells after processed with cisplatin. The slices were subjected to double immunofluorescence staining under the same conditions. β-actin, TGM2 and TGF-β2 polyclonal antibodies (1:300) were used to stain fixed cell slices, followed by incubation with anti-rabbit FITC (Fluorescein isothiocyanate isomer) secondary antibody (1:1,000. Sigma-Aldrich. Merck KGaA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eSW480 cell lines and human tissue samples were lysed with cell lysis buffer containing 1 ml of phenylmethylsulfonyl fluoride. After centrifugation at 12,000 rpm for 15 min, the supernatant was mixed with 5X buffer (SDS-PAGE protein loading buffer) and heated for 10 min. Then, 30\u0026micro;g of total protein was separated using 10% SDS-PAGE protein loading buffer. After blocking with 5% skim milk membrane for 1 hour, the protein was incubated with β-actin, TGM2, and TGF-β2 polyclonal antibodies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eContinuous variables were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical analysis was performed using SPSS software, version 19.0. Group comparisons were performed using t-test, while one-way analysis of variance (ANOVA) was used for intergroup comparisons between all treatment groups and the untreated control group, followed by Dunnett's post hoc test. Differences were considered statistically significant at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eExpression analysis of TGM2 and TGF-β2\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B, the expression levels of TGF-β2 and TGM2 in tumor tissues were significantly higher than those in normal tissues (Welch's t-test, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In addition, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D, the expression levels of TGF-β2 and TGM2 were correlated with TNM stage and tumor maximum diameter (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). As shown in Table\u0026nbsp;1, there was no significant difference in clinical characteristics between patients with high and low expression of TGF-β2.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;1. Chi-square test for clinical characteristics between high and low TGF-β2 expression groups.\u003c/p\u003e\u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eTGF-β\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLow Expression\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHigh Expression\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge(y)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.184\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003egender\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003epathologic T\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e0.043\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003epathologic N\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003epathologic M\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.099\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003elymphatic invasion\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.088\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003elongest dimension(cm)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.009\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eStatistical analysis was based on chi-square test (P\u0026thinsp;\u0026lt;\u0026thinsp;=\u0026thinsp;0.01).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eKaplan-Meier survival analysis\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, in the survival analysis of TGF-β2 and TGM2, patients with high expression had lower overall survival (OS) and disease specific survival (DSS) than those with low expression. The results above indicate that high expression of TGF-β2 and TGM2 has a negative impact on patient prognosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eHE staining\u003c/h2\u003e \u003cp\u003eTissue biopsy is the gold standard for diagnosing tumors, and the most commonly used method is HE staining. The 30 tissue samples we collected were confirmed to be colon cancer tissue by HE staining. HE staining results showed that the cells in adjacent normal tissues were neatly arranged, without swelling and with clear structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), while the tumor tissues showed glandular-like arrangement, with moderate dysplasia and stromal fibrosis, and obvious changes in histological morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry staining\u003c/h2\u003e \u003cp\u003eImmunohistochemistry staining is used to determine the location and relative content of antigens in tissues or cells. The expression level of TGF-β2 in CNCtissues was higher than that in adjacent normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). TGF-β2 in adjacent normal tissues showed negative/weak positive expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Positive expression of TGF-β2 was brown or yellow, mainly concentrated in the nucleus, cytoplasm, and a small amount in the cell membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The expression level of TGM2 in colon cancer tissues was higher than that in adjacent normal tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eThe expression level of TGF-β2 in tumor tissue was significantly higher than that in adjacent normal tissue (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eMTT assay (cisplatin)\u003c/h2\u003e \u003cp\u003eWe treated SW480 cells with different concentrations of cisplatin to detect cell viability. The results showed that the survival rate of SW480 cells treated with cisplatin for 24 hours was decreased compared to untreated SW480 cells (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eEffect of cisplatin on TGF-β2 expression in SW480 cells\u003c/h2\u003e \u003cp\u003eAs shown in the figure, the fluorescence intensity of TGF-β2 in SW480 cells treated with 1 mg/L and 2 mg/L cisplatin was decreased compared to untreated SW480 cells, mainly concentrated in the nucleus and cytoplasm (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-F). The cell structure also underwent significant changes after cisplatin treatment. DAPI (4,6-Diamidino-2-phenylindole), a fluorescent dye that can strongly bind to DNA, was used.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eExpression levels of TGM2 and TGF-β2 after treatment with TGM2 inhibitor\u003c/h2\u003e \u003cp\u003eThe fluorescence intensity of TGM2 in SW480 cells without TGM2 inhibitor treatment was stronger than that in cells treated with TGM2 inhibitor (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-D). The TGM2 inhibitor used was MDC (Dansylcadaverine) at a concentration of 150 \u0026micro;mol/L. After treatment with the TGM2 inhibitor, the fluorescence intensity of TGF-β2 in SW480 cells was weaker than that in untreated cells (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA-D). These results indicate a close correlation between the expression levels of TGM2 and TGF-β2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eAfter analyzing TCGA data, we discovered that the gene expression levels of TGM2 and TGF-β2 were significantly higher in CNC tumor tissue than in normal tissue. Additionally, high expression of TGM2 and TGF-β2 was linked to a worse prognosis, as evidenced by lower overall survival (OS) time and shorter disease specific survival time (DSS) based on Kaplan-Meier survival analysis. This association was likely due to the correlation between the expression levels of TGF-β2 and TGM2 with tumor TNM stage and maximum diameter (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), which ultimately led to a worse prognosis for CNC patients.\u003c/p\u003e \u003cp\u003eTGM2 is a multifunctional protein that participates in numerous cellular processes, such as apoptosis, inflammation, cell proliferation, and signal transduction (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). In terms of cancer development, high expression of TGM2 is linked to cancer progression and resistance (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). TGM2 and TGF-β2 both promote epithelial-mesenchymal transition (EMT) of tumor cells, which is a driving force of tumor metastasis and is linked to tumor cell invasiveness (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Furthermore, TGM2 can activate NF-κB and TGF-β2 can induce lipid droplet formation to induce EMT (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). KANG et al. discovered that high expression of TGM2 promoted infiltration and metastasis of CNC, and knocking out TGM2 inhibited tumor cell growth (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Therefore, the regulatory role of TGM2 is crucial for CNC, and given that the expression level of TGM2 in tumor tissue is much higher than in normal tissue, it is a promising therapeutic target for CNC.\u003c/p\u003e \u003cp\u003eTGF-β2 belongs to the TGF-β superfamily and participates in the regulation of human metabolism, cell growth, and disease development (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). TGF-β2 also promotes cancer progression (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) and is associated with poor prognosis in patients. The TGF-β superfamily has a positive effect on TGM2 expression (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e), and TGM2 and TGF-β2 also have a close mutual regulatory relationship.\u003c/p\u003e \u003cp\u003eOur experimental data confirmed the correlation between TGM2 and TGF-β2, and we found that inhibiting TGM2 expression resulted in decreased expression of TGF-β2. We believe that inhibiting the expression of TGM2 may inhibit the expression of TGF-β2, thereby further affecting the occurrence and development of CNC. Additionally, cisplatin was found to inhibit the expression of TGF-β2, indicating that inhibiting TGM2 could be an effective treatment method for CNC. Preliminary results have shown promise in anti-tumor therapy targeting TGM2 (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur analysis of TCGA data revealed a significant upregulation of TGM2 and TGF-β2 expression levels in CNC tumor tissue compared to normal tissue. Moreover, patients with high expression of TGM2 and TGF-β2 exhibited worse prognosis, indicating their involvement in the progression of CNC. Our results also suggest that TGM2 and TGF-β2 expression levels were correlated with TNM stage and maximum diameter of the tumor (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), which could affect the prognosis of the patient. TGM2 and TGF-β2 were found to promote tumor metastasis by activating EMT, making TGM2 an ideal therapeutic target for CNC. These findings have important clinical significance for the treatment.\u003c/p\u003e \u003cp\u003eHowever, our study has some limitations, including the absence of further clinical trials and insufficient sample size to account for accidental errors. We plan to address these limitations in future studies. Overall, our findings have important clinical implications for the treatment of CNC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThanks to all the patients involved in this study, department of Clinical Pharmacy, School of Pharmaceutical Sciences, Jilin University, department of Gastroenterology, the First Hospital of Jilin University, department of Thoracic surgery, China-Japan Union Hospital of Jilin University.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHai-cheng Gao, Yue Wang and Lei Shi contributed to the conception of the study, Zhe Lv and performed the Bioinformatics analysis and experiment, Lei Shi and Yue Wang contributed significantly to analysis and manuscript preparation, Zhi Hua Cheng performed the data analyses, Lei Shi wrote the manuscript, RiFeng Lu helped perform the analysis with constructive discussions.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Jilin University Fund Support (grant number 4150102000049,415010300074), Changchun Science and Technology Bureau (19SS015), the project comes from the Department of science and technology of Jilin Province (grant number 20200201459jc), Jilin Province direct health special project (grant number JLSWSRCZX2020-008), Jilin Provincial Finance Department (grant number JLSWSRCZX2020-083), Education Department of Jilin Province (grant number JJKH20211209KJ).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used and/or analysed in this study were downloaded from the Cancer Genome Atlas public database, available at: https://portal.gdc.cancer.gov/.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis trial was conducted in accordance with the principles outlined in the World Medical Association Declaration of Helsinki for experiments involving human subjects, as revised in 2013. This study was approved by the Research Ethics Committee of China-Japan Union Hospital of Jilin University. Patients gave written informed consent before specimen collection, and the experimental data and personal information they provided were kept completely confidential.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no economic and non-economic competitive interest associated with research.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026sup1; (Department of Clinical Pharmacy, School of Pharmaceutical Sciences, Jilin University, Changchun. Department of Gastroenterology, the First Hospital of Jilin University, Changchun, China. Department of Thoracic surgery, China-Japan Union Hospital of Jilin University, Changchun, China.)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBaidoun F, Elshiwy K, Elkeraie Y, Merjaneh Z, Khoudari G, Sarmini MT, Gad M, Al-Husseini M, Saad A. Colorectal Cancer Epidemiology: Recent Trends and Impact on Outcomes. Curr Drug Targets. 2021;22(9):998-1009. doi: 10.2174/1389450121999201117115717. PMID: 33208072.\u003c/li\u003e\n\u003cli\u003eLi H, Li L, Huang X, Li Y, Zou T, Zhuo X, Chen Y, Liu Y, Tang Y. Radiotherapy-induced dysphagia and its impact on quality of life in patients with nasopharyngeal carcinoma. Strahlenther Onkol. 2019 Jun;195(6):457-467. English. doi: 10.1007/s00066-018-01421-6. Epub 2019 Jan 28. PMID: 30689027.\u003c/li\u003e\n\u003cli\u003eDekker E, Tanis PJ, Vleugels JLA, Kasi PM, Wallace MB. Colorectal cancer. Lancet. 2019 Oct 19;394(10207):1467-1480. doi: 10.1016/S0140-6736(19)32319-0. PMID: 31631858.\u003c/li\u003e\n\u003cli\u003eEckert RL, Kaartinen MT, Nurminskaya M, Belkin AM, Colak G, Johnson GV, Mehta K. Transglutaminase regulation of cell function. Physiol Rev. 2014 Apr;94(2):383-417. doi: 10.1152/physrev.00019.2013. PMID: 24692352; PMCID: PMC4044299.\u003c/li\u003e\n\u003cli\u003eKang S, Oh SC, Min BW, Lee DH. Transglutaminase 2 Regulates Self-renewal and Stem Cell Marker of Human Colorectal Cancer Stem Cells. Anticancer Res. 2018 Feb;38(2):787-794. doi: 10.21873/anticanres.12285. PMID: 29374703.\u003c/li\u003e\n\u003cli\u003eShan H, Zhou X, Chen C. MicroRNA‑214 suppresses the viability, migration and invasion of human colorectal carcinoma cells via targeting transglutaminase 2. Mol Med Rep. 2019 Aug;20(2):1459-1467. doi: 10.3892/mmr.2019.10325. Epub 2019 Jun 3. PMID: 31173203; PMCID: PMC6625444.\u003c/li\u003e\n\u003cli\u003eMorikawa M, Derynck R, Miyazono K. TGF-\u0026beta; and the TGF-\u0026beta; Family: Context-Dependent Roles in Cell and Tissue Physiology. Cold Spring Harb Perspect Biol. 2016 May 2;8(5):a021873. doi: 10.1101/cshperspect.a021873. PMID: 27141051; PMCID: PMC4852809.\u003c/li\u003e\n\u003cli\u003eSyed V. TGF-\u0026beta; Signaling in Cancer. J Cell Biochem. 2016 Jun;117(6):1279-87. doi: 10.1002/jcb.25496. Epub 2016 Feb 11. PMID: 26774024.\u003c/li\u003e\n\u003cli\u003eBae GH, Kim YS, Park JY, Lee M, Lee SK, Kim JC, Kim JG, Shin YJ, Lee H, Kim SY, Bae YS, Zabel BA, Kim HS, Bae YS. Unique characteristics of lung-resident neutrophils are maintained by PGE2/PKA/Tgm2-mediated signaling. Blood. 2022 Aug 25;140(8):889-899. doi: 10.1182/blood.2021014283. PMID: 35679477; PMCID: PMC9412003.\u003c/li\u003e\n\u003cli\u003eMaggio N, Sellitti S, Capano CP, Papa M. Tissue-transglutaminase in rat and human brain: light and electron immunocytochemical analysis and in situ hybridization study. Brain Res Bull. 2001 Oct-Nov 1;56(3-4):173-82. doi: 10.1016/s0361-9230(01)00649-9. PMID: 11719248.\u003c/li\u003e\n\u003cli\u003eLiu H, Jiang B. Let-7a-5p represses proliferation, migration, invasion and epithelial-mesenchymal transition by targeting Smad2 in TGF-b2-induced human lens epithelial cells. J Biosci. 2020;45:59. PMID: 32345785.\u003c/li\u003e\n\u003cli\u003eShrestha R, Tatsukawa H, Shrestha R, Ishibashi N, Matsuura T, Kagechika H, Kose S, Hitomi K, Imamoto N, Kojima S. Molecular mechanism by which acyclic retinoid induces nuclear localization of transglutaminase 2 in human hepatocellular carcinoma cells. Cell Death Dis. 2015 Dec 3;6(12):e2002. doi: 10.1038/cddis.2015.339. PMID: 26633708; PMCID: PMC4720877.\u003c/li\u003e\n\u003cli\u003eKang JH, Lee SH, Kim SY. Discovery of a novel target for renal cell carcinoma: transglutaminase 2. Cell Death Dis. 2016 Apr 21;7(4):e2200. doi: 10.1038/cddis.2016.99. PMID: 27100894; PMCID: PMC4855678.\u003c/li\u003e\n\u003cli\u003eDas V, Bhattacharya S, Chikkaputtaiah C, Hazra S, Pal M. The basics of epithelial-mesenchymal transition (EMT): A study from a structure, dynamics, and functional perspective. J Cell Physiol. 2019 Sep;234(9):14535-14555. doi: 10.1002/jcp.28160. Epub 2019 Feb 5. PMID: 30723913.\u003c/li\u003e\n\u003cli\u003eCorbet C, Bastien E, Santiago de Jesus JP, Dierge E, Martherus R, Vander Linden C, Doix B, Degavre C, Guilbaud C, Petit L, Michiels C, Dessy C, Larondelle Y, Feron O. TGF\u0026beta;2-induced formation of lipid droplets supports acidosis-driven EMT and the metastatic spreading of cancer cells. Nat Commun. 2020 Jan 23;11(1):454. doi: 10.1038/s41467-019-14262-3. PMID: 31974393; PMCID: PMC6978517.\u003c/li\u003e\n\u003cli\u003eTakahashi H, Alves CRR, Stanford KI, Middelbeek RJW, Nigro P, Ryan RE, Xue R, Sakaguchi M, Lynes MD, So K, Mul JD, Lee MY, Balan E, Pan H, Dreyfuss JM, Hirshman MF, Azhar M, Hannukainen JC, Nuutila P, Kalliokoski KK, Nielsen S, Pedersen BK, Kahn CR, Tseng YH, Goodyear LJ. TGF-\u0026beta;2 is an exercise-induced adipokine that regulates glucose and fatty acid metabolism. Nat Metab. 2019 Feb;1(2):291-303. doi: 10.1038/s42255-018-0030-7. Epub 2019 Feb 11. PMID: 31032475; PMCID: PMC6481955.\u003c/li\u003e\n\u003cli\u003eDropmann A, Dooley S, Dewidar B, Hammad S, Dediulia T, Werle J, Hartwig V, Ghafoory S, Woelfl S, Korhonen H, Janicot M, Wosikowski K, Itzel T, Teufel A, Schuppan D, Stojanovic A, Cerwenka A, Nittka S, Piiper A, Gaiser T, Beraza N, Milkiewicz M, Milkiewicz P, Brain JG, Jones DEJ, Weiss TS, Zanger UM, Ebert M, Meindl-Beinker NM. TGF-\u0026beta;2 silencing to target biliary-derived liver diseases. Gut. 2020 Sep;69(9):1677-1690. doi: 10.1136/gutjnl-2019-319091. Epub 2020 Jan 28. PMID: 31992593; PMCID: PMC7456737.\u003c/li\u003e\n\u003cli\u003eSun Y, Xiong L, Wang X, Wang L, Chen B, Huang J, Huang M, Chen J, Wu J, Huang S, Liu Y. Autophagy inhibition attenuates TGF-\u0026beta;2-induced epithelial-mesenchymal transition in lens epithelial cells. Life Sci. 2021 Jan 15;265:118741. doi: 10.1016/j.lfs.2020.118741. Epub 2020 Nov 10. PMID: 33181173.\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":"colon cancer，TGM2，TGF-β2，immunohistochemistry","lastPublishedDoi":"10.21203/rs.3.rs-2781254/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2781254/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eColon cancer (CNC) ranks third in incidence and second in mortality among all cancers worldwide. Unfortunately, the available treatment options for CNC have limited effectiveness in improving patients' prognosis, and the pathogenesis of CNC remains poorly understood. Transglutaminase 2 (TGM2) and TGF-β2 both play a positive role in regulating cancer progression, promoting tumor progression and metastasis. However, the mutual relationship between TGM2 and TGF-β2 has not been well studied. Our analysis of transcriptome data from CNC patients on the TCGA website showed that the expression levels of TGF-β2 and TGM2 were related to TNM staging and tumor size (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and high expression of TGF-β2 and TGM2 was associated with a worse prognosis (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). To validate these findings, we performed experiments using collected tumor tissue specimens from CNC patients, which demonstrated that inhibiting TGM2 could reduce the expression of TGF-β2 and improve the prognosis of CNC patients. Therefore, targeting TGM2 and TGF-β2 may become feasible therapeutic targets for CNC, and our study has provided directions for the development of CNC treatment drugs.\u003c/p\u003e","manuscriptTitle":"The synergistic effect of TGM2 and TGFβ2 on the prognosis of colon cancer patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-05-09 16:09:27","doi":"10.21203/rs.3.rs-2781254/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":"34a598a7-9600-49c9-8f09-0b4a4e238226","owner":[],"postedDate":"May 9th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-12-31T07:59:18+00:00","versionOfRecord":[],"versionCreatedAt":"2023-05-09 16:09:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2781254","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2781254","identity":"rs-2781254","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}