Research on the mechanism by which USP18 promotes colorectal cancer progression through stabilizing KRT16 expression

Research on the mechanism by which USP18 promotes colorectal cancer progression through stabilizing KRT16 expression | 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 Research on the mechanism by which USP18 promotes colorectal cancer progression through stabilizing KRT16 expression LIN ZHANG, XIN LI, HONGJIE YANG, ZHIQIN GUO, XIANGYI CHEN, WANXIN WU This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8191897/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 This study aims to investigate the effects of Ubiquitin-specific peptidase 18 (USP18) and Keratin16 (KRT16) on the progression of colorectal cancer and their possible mechanism of action. Methods The downstream target protein of USP18 was identified using Co-immunoprecipitation(Co-IP) and mass spectrometry. The clinical significance of KRT16 was validated via bioinformatics analysis of the TCGA database. Immunohistochemical staining detected USP18 and KRT16 expression in CRC tissues, and their relationship with clinicopathological features was analyzed. In vitro functional assays, including Western Blot, scratch tests, and CCK-8 assays, were performed using USP18 overexpression and KRT16 interference plasmids. Results KRT16 was identified as a downstream target of USP18, and USP18 overexpression was shown to deubiquitinate KRT16. Bioinformatics analysis revealed that KRT16 was highly expressed in CRC (P < 0.001), exhibited significant prognostic value (log-rank P = 0.029) and high diagnostic accuracy (AUC = 0.899). A positive correlation was found between KRT16 and USP18 expression (R = 0.308, P < 0.001). Immunohistochemistry confirmed that both KRT16 and USP18 were significantly upregulated in CRC tissues, with KRT16 expression correlating with T stage (P = 0.008). In vitro, KRT16 knockdown inhibited CRC cell proliferation and migration. Importantly, USP18 overexpression enhanced proliferation and migration, which was rescued by KRT16 knockdown. Conclusion KRT16 can be used as one of the markers of colorectal cancer, and USP18 may be involved in the occurrence and development of colorectal cancer by regulating the expression of KRT16. Colorectal cancer USP18 KRT16 Proliferation Migration Figures Figure 1 Figure 2 Figure 3 1 Introduction Colorectal cancer (CRC) is the third leading cause of cancer-related deaths globally[ 1 ]. Early diagnosis and treatment can significantly improve survival rates[ 2 ]. Despite advancements in understanding the etiology, pathogenesis, and epidemiology of CRC, its molecular mechanisms remain incompletely elucidated. Additionally, the increasing incidence of CRC in younger populations and distant metastasis remain major contributors to patient mortality[ 3 ]. Therefore, investigating the underlying mechanisms of CRC progression and identifying novel therapeutic targets are crucial for improving patient outcomes. Our previous studies demonstrated that USP18 promotes colorectal cancer cell proliferation and may serve as a potential diagnostic biomarker[ 4 ]. USP18, a deubiquitinating enzyme, is upregulated in various cancers and functions to inhibit apoptosis and promote tumorigenesis[ 5 – 6 ]. However, its downstream regulatory targets in CRC remain unclear. Keratins, intermediate filament proteins, maintain structural integrity in epithelial cells, regulate cell size, determine organelle positioning, and influence protein translation and cell polarity[ 7 – 8 ]. Fifty-four keratins have been identified in human epithelial cells, including 28 type I (acidic forms, K9–K28) and 26 type II (basic forms, K1–K8 and K71–K74) proteins[ 9 ]. Studies suggest that keratins influence tumorigenesis and metastasis. In our in-depth investigation of the downstream regulatory mechanisms of USP18, we have noted that the keratin family member KRT16, due to its aberrant expression in various epithelial-derived tumors and close association with malignant progression, has emerged as a noteworthy potential target. For instance, Elevated KRT16 mRNA expression is associated with reduced recurrence-free survival (RFS) in metastatic breast cancer[ 10 ], and KRT16 is upregulated in oral squamous cell carcinoma (OSCC) cell lines and tumor tissues[ 11 ]. Understanding the mechanistic role of KRT16 in tumorigenesis and its functional impact is essential for both basic research and therapeutic targeting. In this study, we employed mass spectrometry, TCGA database analysis, clinical tumor tissues, and in vitro experiments to investigate the interaction between USP18 and KRT16. We aimed to determine whether KRT16 serves as a biomarker for CRC and whether USP18 regulates KRT16 expression to drive CRC progression. Our findings provide a theoretical foundation for identifying novel therapeutic targets in CRC. 2 Materials and Methods 2.1 Clinical specimens One hundred colorectal cancer tissue specimens and paired clinical-pathological data were collected from patients treated at Jiaxing First Hospital between January 2019 and December 2020 (67 males, 33 females). All cases were pathologically confirmed, with no prior radiotherapy or chemotherapy. This study was approved by the Ethics Committee of Jiaxing First Hospital (2024-LY-603). Clinical trial number: not applicable. 2.2 Cells and reagents The human colon cancer cell line SW480 was purchased from Procell Life Science & Technology Co., Ltd. Fetal bovine serum (40131ES76), reverse transcription kits (11141ES), SYBR Green Master Mix (11184ES), and CCK-8 (40203ES76) were obtained from Yeasen Biotechnology. RPMI-1640 medium (MA0215) was purchased from Meilun Biotechnology. Penicillin-streptomycin solution (C0222), IP lysis buffer (P0013), RIPA buffer (P0013B), and Lipo8000™ transfection reagent (C0533) were acquired from Beyotime Biotechnology(Shanghai,China). The Real EnVision Detection System K5007 (DAKO) was used for immunohistochemistry(Copenhagen, Denmark). MG132 (S2619), Protein A/G magnetic beads (B23201), and Anti-Flag magnetic beads (B26101) were purchased from Selleck. Antibodies against USP18 (A16739), KRT16 (A7493), PCNA (A0264), Cyclin B1 (A16038), Cyclin D1 (A11022), and β-actin (AC004) were purchased from ABclonal(Wuhan,China). IRDye 800CW goat anti-rabbit (926-32211) and anti-mouse (926-32210) secondary antibodies were obtained from LI-COR. Trizol reagent (15596026CN) and BCA protein assay kits were purchased from Thermo Fisher Scientific. 2.3 Cell culture and transfection SW480 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C under 5% CO₂.SW480 cells were seeded into 6-well plates 24 hours prior to transfection to achieve 70–80% confluency. Plasmid DNA was diluted in serum-free RPMI-1640 medium and mixed with Lipo8000™ transfection reagent. The mixture was added dropwise to cells and incubated for 48 hours. 2.4 Western blotting Proteins were extracted using RIPA buffer and quantified via BCA assay. Samples (20 µg/lane) were separated by SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk. Membranes were incubated with primary antibodies (USP18: 1:500; KRT16: 1:1000; PCNA: 1:1000; Cyclin B1: 1:1000; Cyclin D1: 1:1000; β-actin: 1:3000) overnight at 4°C. After incubation with IRDye 800CW secondary antibodies (1:10,000), protein bands were visualized using an Odyssey DLx Imaging System (LI-COR) and analyzed with ImageJ. 2.5 Co-Immunoprecipitation (Co-IP) Upon transfection with USP18, the proteasome inhibitor MG132 (MCE, Cat# HY-13259) was added 6 hours prior to cell collection. After harvesting, total proteins were extracted using IP lysis buffer (Beyotime, Cat# P0013). A small portion of the total protein was reserved as the Input control, while the remaining protein samples were equally divided and incubated overnight at 4°C with rotation with either Protein A/G immunoprecipitation magnetic beads (MCE, Cat# HY-K0202) pre-coated with IgG isotype control antibody (CST, Cat# 3900S) or Anti-Flag magnetic beads (MCE, Cat# HY-K0207). The next day, samples were denatured with 1×Loading Buffer (Beyotime, Cat# P0015) at 95°C for 10 minutes, followed by protein interaction analysis via Western Blot. 2.6 Immunofluorescence (IF) Seeding and culturing adherent cells on sterile glass coverslips until they reach 50–70% confluence. The cells are then washed with phosphate-buffered saline (PBS) and fixed, typically with 4% paraformaldehyde, to preserve cellular architecture. Following fixation, cells are permeabilized with a detergent solution such as 0.1% Triton X-100 to allow antibody access to intracellular targets. Non-specific binding sites are subsequently blocked using a solution of bovine serum albumin (BSA). The cells are then incubated with a primary antibody specific to the protein of interest, diluted in blocking buffer, after which unbound antibody is removed by washing. A fluorophore-conjugated secondary antibody, directed against the host species of the primary antibody, is applied to visualize the target. Finally, the coverslips are mounted onto microscope slides using an anti-fade mounting medium containing a nuclear counterstain like DAPI, and the samples are visualized using fluorescence. 2.7 Immunohistochemistry (IHC) Tissue sections were deparaffinized in xylene and rehydrated through a graded ethanol series. Antigen retrieval was performed by heating the sections in citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide for 15 minutes. Subsequently, the sections were incubated overnight at 4°C with primary antibodies against USP18 (1:800) and KRT16 (1:400). For the negative control, the primary antibody was replaced with PBS. After primary antibody incubation, the sections were treated with an enhanced enzyme-labeled goat anti-mouse/rabbit IgG polymer (EnVision™ system)—a method that conjugates the secondary antibody and enzyme into a polymer (i.e., EnVision) to amplify the signal. Color development was achieved using a freshly prepared DAB substrate solution. Finally, the sections were counterstained with hematoxylin, dehydrated, and mounted with neutral balsam. IHC results were assessed based on the proportion of positive cells and the staining intensity. The presence of brown-yellow or brown granules in the cytoplasm was considered positive staining. All slides were independently evaluated by two senior pathologists under double-blind conditions. Five random fields per section were examined under a 400× light microscope and scored according to the following criteria:percentage of positive cells: 0% (0 points); ≤10% (1 point); 11%–50% (2 points); 51%–70% (3 points); ≥71% (4 points). staining intensity: No staining (0 points); pale yellow (1 point); brown-yellow (2 points); brown (3 points).The final staining score (ranging from 0 to 12) was calculated by multiplying the score for the percentage of positive cells by the staining intensity score. A final score of ≥ 6 was defined as high expression, and a score of < 6 was defined as low expression. 2.8 Wound healing assay SW480 cells were transfected for 48 hours, and a scratch was created using a 200 µl pipette tip. Cells were washed with PBS and cultured in serum-free medium. Migration was monitored at 0 and 24 hours using an inverted microscope. Wound closure was quantified with ImageJ. 2.9 Transwell migration assay Cells in the logarithmic growth phase were trypsinized, resuspended in serum-free medium, and adjusted to the appropriate concentration. A 100–200 µL cell suspension was added to the upper chamber, which was then inserted into a 24-well plate. The plate was incubated at 37°C with 5% CO₂ for 24 h. After incubation, the Transwell chamber was carefully removed, and non-migrated cells on the upper surface of the membrane were gently wiped off using a moistened cotton swab. The chamber was fixed in 4% paraformaldehyde for 15–30 min, followed by staining with 0.1% crystal violet for 10–30 min. Excess stain was removed by washing with PBS until no floating color remained, and the membrane was air-dried. Migrated cells on the lower surface of the membrane were counted under a microscope (100× or 200× magnification) by randomly selecting 5–12 fields of view. 2.10 Cell Counting Kit-8 (CCK-8) assay Transfected SW480 cells (2,000 cells/well) were seeded into 96-well plates. Cell viability was assessed at 24, 48, 72, and 96 hours using CCK-8 reagent. Absorbance at 450 nm was measured. 2.11 Quantitative Real-time Polymerase Chain Reaction (qPCR) analysis Total RNA was extracted with Trizol, reverse-transcribed into cDNA, and amplified using SYBR Green Master Mix. KRT16 expression was normalized to β-Actin (ACTB) and calculated via the 2 − ΔΔCt method. Primer sequences: Homo KRT16: Forward: 5′-AAAGGAACCGCCCCAAATCT-3′ Reverse: 5′-GTATCTCTGTGCCCGGACTG-3′ Homo ACTB: Forward: 5′-GAGAAAATCTGGCACCACACC-3′ Reverse: 5′-GGATAGCACAGCCTGGATAGCAA-3′ 2.12 Statistical Analysis Data are presented as mean ± SD. Comparisons between groups were analyzed using Student’s t-test (normally distributed data) or Wilcoxon rank-sum test (non-normal data). Spearman’s correlation and log-rank tests were used for survival analysis. All analyses were performed using GraphPad Prism 10.0 and Xiantao Academic ( https://www.xiantaozi.com/ ). 3 Results 3.1 KRT16 is a potential downstream target of USP18 Based on our mass spectrometry analysis of USP18-interacting proteins, we identified keratin 16 (KRT16) as a strong candidate binding partner (Fig. 1 A). To explore the functional consequence of this interaction, we first established a robust USP18 overexpression system, which resulted in a dramatic increase in USP18 mRNA (Fig. 1 B). Notably, this forced expression of USP18 led to a significant upregulation of KRT16 protein levels (Fig. 1 C), suggesting a potential regulatory relationship. We further confirmed the physical association between these two proteins by demonstrating their direct interaction under physiological conditions using an endogenous co-immunoprecipitation assay (Fig. 1 D). Given the known deubiquitinating function of USP18, we hypothesized that it might stabilize KRT16 by modulating its ubiquitination. Consistent with this, overexpression of USP18 substantially reduced the ubiquitination level of KRT16 (Fig. 1 E). Conversely, knockdown of USP18 effectively enhanced KRT16 ubiquitination (Fig. 1 F), providing compelling evidence that USP18 negatively regulates KRT16's ubiquitin-mediated degradation. Finally, immunofluorescence microscopy visually confirmed the functional interaction between USP18 and KRT16, revealing their distinct colocalization within cells (Fig. 1 G). 3.2 KRT16 expression and prognostic significance in CRC Based on the comprehensive bioinformatics analysis of the TCGA database, we observed a significant overexpression of KRT16 in colorectal cancer (CRC) tissues relative to normal counterparts ( P < 0.001, Fig. 2 A). A positive correlation was further identified between the expression levels of KRT16 and USP18 ( R = 0.308, P < 0.001, Fig. 2 B). The diagnostic potential of KRT16 for CRC was underscored by receiver operating characteristic (ROC) curve analysis, which yielded a high area under the curve(AUC) of 0.899 (Fig. 2 C). From a prognostic standpoint, Kaplan–Meier survival analysis revealed that elevated KRT16 expression was associated with markedly shorter overall survival, corresponding to a 1.5-fold increase in mortality risk (Hazard Ratio [HR] = 1.55, P = 0.029, Fig. 2 D). These findings were further corroborated by our validation cohort. Western blot analysis of 14 paired clinical samples from Jiaxing First Hospital confirmed a significant upregulation of KRT16 protein in CRC tissues compared to adjacent normal mucosa ( P < 0.05, Fig. 2 E). Consistent with this, immunohistochemical staining of a larger series of 100 paraffin-embedded CRC specimens demonstrated significantly higher protein levels of both KRT16 and USP18 in cancerous tissues ( P < 0.05, Fig. 2 F). Furthermore, clinicopathological analysis indicated that high KRT16 expression was significantly associated with advanced T stage ( P = 0.008) and showed a strong positive correlation with USP18 expression ( P < 0.001, Table 1 ), reinforcing its clinical relevance in CRC progression. Table 1 Expression of KRT16 in 100 colorectal cancer tissues and its relationship with clinicopathological characteristics [n (%)] Characteristics Total (n = 100) KRT16 χ² P Low High Gender, n(%) χ²=0.86 0.353 Male 67 (67.00) 24 (61.54) 43 (70.49) Female 33 (33.00) 15 (38.46) 18 (29.51) Ages, n(%) χ²=0.20 0.657 < 60 36 (36.00) 13 (33.33) 23 (37.70) ≥ 60 64 (64.00) 26 (66.67) 38 (62.30) Location, n(%) χ²=1.57 0.210 Colon 59 (59.00) 20 (51.28) 39 (63.93) Rectum 41 (41.00) 19 (48.72) 22 (36.07) Diameter, n(%) χ²=0.05 0.828 < 5cm 73 (73.00) 28 (71.79) 45 (73.77) ≥ 5cm 27 (27.00) 11 (28.21) 16 (26.23) Tumor type, n(%) χ²=0.00 1.000 Other 3 (3.00) 1 (2.56) 2 (3.28) Adenocarcinoma 97 (97.00) 38 (97.44) 59 (96.72) Differentiation grade, n(%) - 0.076 Low 5 (5.00) 1 (2.56) 4 (6.56) High 3 (3.00) 3 (7.69) 0 (0.00) Moderate 92 (92.00) 35 (89.74) 57 (93.44) T stage, n(%) χ²=7.06 0.008 T1 ~ T2 18 (18.00) 12 (30.77) 6 (9.84) T3 ~ T4 82 (82.00) 27 (69.23) 55 (90.16) N stage, n(%) χ²=1.18 0.277 N0 60 (60.00) 26 (66.67) 34 (55.74) N1 ~ N2 40 (40.00) 13 (33.33) 27 (44.26) M stage, n(%) χ²=0.00 1.000 M0 96 (96.00) 37 (94.87) 59 (96.72) M1 4 (4.00) 2 (5.13) 2 (3.28) Pathological stage, n(%) χ²=1.18 0.277 Ⅰ~Ⅱ 60 (60.00) 26 (66.67) 34 (55.74) Ⅲ~Ⅳ 40 (40.00) 13 (33.33) 27 (44.26) USP18, n(%) χ²=35.55 < .001 Low 36 (36.00) 28 (71.79) 8 (13.11) High 64 (64.00) 11 (28.21) 53 (86.89) 3.3 KRT16 knockdown attenuates USP18-driven CRC proliferation and migration To investigate the functional role of KRT16, we transfected CRC cells with sh-KRT16 plasmids. qPCR confirmed efficient knockdown (P < 0.001, Fig. 3 A). Western blot analysis of proliferation markers (PCNA, Cyclin B1, and Cyclin D1) revealed that KRT16 silencing significantly suppressed their protein expression levels compared to the control group (all P < 0.05, Fig. 3 B). Scratch wound healing and Transwell assays further demonstrated that KRT16 knockdown inhibited the migratory capacity of colon cancer cells (P < 0.05, Fig. 3 C, D). Subsequently, cells were co-transfected with sh-KRT16 and USP18 overexpression plasmids. Western blot analysis showed that while KRT16 interference reduced the expression of proliferation markers, USP18 overexpression enhanced their expression. Notably, in the co-transfection group, the USP18-induced upregulation of these markers was significantly attenuated (Fig. 3 E). CCK-8 assays corroborated these findings: KRT16 knockdown suppressed cell proliferation, USP18 overexpression promoted it, and the pro-proliferative effect of USP18 was partially reversed in co-transfected cells (Fig. 3 F). These results suggest that USP18 likely regulates proliferative capacity through KRT16. Similarly, scratch and Transwell assays confirmed that KRT16 knockdown impaired cell migration, USP18 overexpression enhanced it, and co-transfection counteracted the promigratory effect of USP18 (P < 0.05, Fig. 3 G,H). Collectively, these data indicate that USP18 promotes colon cancer cell proliferation and migration by modulating KRT16. 4 Discussion Colorectal cancer (CRC) is a common malignant tumor of the digestive tract and the third leading cause of cancer-related deaths. Due to its insidious clinical manifestations, CRC is often diagnosed at advanced stages, with high rates of postoperative recurrence and metastasis, resulting in poor prognosis and low 5-year survival rates[ 12 ]. Although treatment options for CRC have expanded in recent years, including surgery, radiotherapy, chemotherapy, and immunotherapy, the heterogeneity and metastatic potential of CRC continue to pose significant challenges. Consequently, the 5-year survival rate for advanced CRC patients remains unsatisfactory. Therefore, elucidating the molecular mechanisms underlying CRC progression and identifying novel biomarkers are critical for improving early diagnosis, prognosis prediction, and targeted therapy. Our previous study demonstrated that elevated USP18 expression is associated with CRC proliferation and metastasis[ 4 ]. However, the precise mechanisms by which USP18 promotes CRC remain unclear. As a deubiquitinating enzyme, USP18 likely exerts its oncogenic effects by stabilizing key regulatory proteins. In this study, we employed co-immunoprecipitation (Co-IP) combined with mass spectrometry to identify potential downstream targets of USP18, ultimately identifying keratin 16 (KRT16) as a candidate. Keratins are well-established tumor markers in clinical practice, implicated in epithelial metaplasia, malignant transformation, and metastasis. Based on their isoelectric points, keratins are classified into acidic type I (CK9–CK20) and basic type II (CK1–CK8) subtypes[ 13 ]. Previous studies have shown that CK8, CK18, and CK19 are overexpressed in CRC tissues compared to adjacent normal tissues and may contribute to CRC progression[ 14 ]. For instance, CK18 upregulation activates the PI3K-AKT pathway and epithelial-mesenchymal transition (EMT), thereby promoting CRC cell proliferation, invasion, and metastasis[ 15 ]. The KRT16 gene, located on chromosome 17q21.23, has been implicated in the pathogenesis of various cancers, including nasopharyngeal carcinoma, breast cancer, and lung adenocarcinoma, where it exhibits oncogenic properties and correlates with poor prognosis[ 16 – 17 ]. However, the role of KRT16 in CRC remains largely unexplored. In this study, we found that KRT16 expression was significantly higher in CRC tissues than in adjacent normal tissues, suggesting its potential diagnostic value. Furthermore, KRT16 protein expression and T stage were independent prognostic factors for CRC patients, with high KRT16 expression associated with shorter overall survival. These findings indicate that KRT16 may play a pivotal role in CRC progression and warrant further clinical validation. Previous studies have reported that KRT16 is overexpressed in lung adenocarcinoma cells, and its knockdown suppresses proliferation, invasion, migration, and EMT[ 18 ]. In this study, we used shRNA-mediated KRT16 knockdown and demonstrated that silencing KRT16 significantly inhibited the proliferative and migratory capacities of CRC cells. Given the positive correlation and direct interaction between USP18 and KRT16 in CRC, we co-transfected USP18 overexpression plasmids with KRT16 shRNA into CRC cells. The results showed that KRT16 knockdown attenuated the tumor-promoting effects of USP18, suggesting that USP18 may drive CRC progression, at least in part, by stabilizing KRT16. In summary, KRT16 is overexpressed in CRC and serves as an independent prognostic marker for poor survival. The USP18-KRT16 axis may represent a promising therapeutic target for CRC intervention. Further studies are needed to validate these findings in larger clinical cohorts and explore the detailed molecular mechanisms underlying USP18-mediated KRT16 stabilization. Declarations Author contributions L.Z. was responsible for study implementation and data collection, and wrote the manuscript. X.C. and X.L. conducted data analysis and statistics. H.Y. and Z.G. provided technical and material support. W.W. supervised the study, reviewed the manuscript, and acquired funding. All authors reviewed the manuscript. Funding Zhejiang Provincial Medical and Health Science and Technology Project (2025KY1584); Jiaxing Key Medical Discipline (2023-ZC-017); Jiaxing Civil Science and Technology Innovation Special Project (2020AY30008) Data availability The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author. Ethics approval The procedures used in this study adhere to the tenets of the Declaration of Helsinki. And all experimental procedures were approved by the Ethics Committee of Jiaxing First Hospital (2024-LY-603). Consent to participate Informed consent was obtained from all individual participants included in the study. Consent for publication Written informed consent was obtained from each participant. Competing interests The authors declare no competing interests. References Sung H., Ferlay J., Siegel R.L., Laversanne M., Soerjomataram I., Jemal A. and Bray F. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 2021;71(3): 209-249. DOI:10.3322/caac.21660. Stark U.A., Frese T., Unverzagt S., Danner D., Unverzagt M. and Herrmann K. What is the effectiveness of various invitation methods to colonoscopy in early detection of colorectal cancer? Protocol of a systematic review. Syst. Rev. 2020;9(1):49. DOI:10.1186/s13643-020-01312-x. Molecular Diagnostics Group of Chinese Society of Laboratory Medicine. 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09:53:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8191897/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8191897/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":97896730,"identity":"4b128beb-1823-4e16-afeb-f1ca8110a211","added_by":"auto","created_at":"2025-12-10 15:36:57","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":411217,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-8191897/v1/9117b826251e1a900cb7ea83.docx"},{"id":97896635,"identity":"013646d7-0259-45ab-aef4-5b2718860c66","added_by":"auto","created_at":"2025-12-10 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18:56:43","extension":"html","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":90583,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8191897/v1/95b160ba5dfe7e2afe06872a.html"},{"id":97734035,"identity":"4218216b-6912-4c28-88ac-572614f0d797","added_by":"auto","created_at":"2025-12-08 18:56:43","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":106585,"visible":true,"origin":"","legend":"\u003cp\u003eKRT16 may be a downstream effector protein of USP18. \u003cstrong\u003e(A)\u003c/strong\u003e Mass spectrometry screening for downstream target proteins of USP18; \u003cstrong\u003e(B) \u003c/strong\u003eValidation of transfection efficiency of USP18 overexpression plasmid by qPCR; \u003cstrong\u003e(C) \u003c/strong\u003eWestern Blot analysis of changes in protein expression of KRT16 and β-actin post-transfection; \u003cstrong\u003e(D) \u003c/strong\u003eWestern Blot analysis of KRT16 expression following Co-IP; \u003cstrong\u003e(E)\u003c/strong\u003e Western Blot analysis of ub expression following Co-IP after transfection of myc-KRT16 and flag-USP18; \u003cstrong\u003e(F)\u003c/strong\u003e Western Blot analysis of ub expression following Co-IP after transfection of myc-KRT16 and flag-shUSP18; \u003cstrong\u003e(G)\u003c/strong\u003e using IF to detect the expression of USP18 and KRT16.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8191897/v1/a6878de4fc5eb5d2b4cb66f8.jpeg"},{"id":97895956,"identity":"b332125a-d70d-4239-b5aa-90ecaea8f269","added_by":"auto","created_at":"2025-12-10 15:35:27","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":144521,"visible":true,"origin":"","legend":"\u003cp\u003eExpression and prognostic significance of KRT16 in colorectal cancer. \u003cstrong\u003e(A) \u003c/strong\u003eAnalysis of KRT16 expression in tumor and adjacent normal tissues using TCGA database; \u003cstrong\u003e(B) \u003c/strong\u003eExamination of the relationship between KRT16 and USP18 expression using TCGA database; \u003cstrong\u003e(C)\u003c/strong\u003e Determination of the ROC value for KRT16 using TCGA database; \u003cstrong\u003e(D)\u003c/strong\u003e Correlation between KRT16 expression levels and patient survival using TCGA database; \u003cstrong\u003e(E) \u003c/strong\u003eWestern Blot analysis of KRT16 and β-actin protein expression in tumor and adjacent normal tissues; \u003cstrong\u003e(F)\u003c/strong\u003e IHC detection of KRT16 and USP18 expression in tumor and adjacent normal tissues using the EnVision method, high magnification.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8191897/v1/2fc4c805c2421cb94a12f4d0.jpeg"},{"id":97734039,"identity":"cac227bd-de4d-4d84-b1e8-9d81e231af74","added_by":"auto","created_at":"2025-12-08 18:56:43","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":167053,"visible":true,"origin":"","legend":"\u003cp\u003eKnockdown of KRT16 inhibits USP18-mediated promotion of proliferation and migration in colon cancer cells. \u003cstrong\u003e(A)\u003c/strong\u003e qPCR assessment of shKRT16 knockdown efficiency; \u003cstrong\u003e(B)\u003c/strong\u003eWestern Blot analysis of protein expression levels of PCNA, Cyclin B1, Cyclin D1, and β-actin; \u003cstrong\u003e(C)\u003c/strong\u003e Scratch assay to evaluate cell migration ability in different groups; \u003cstrong\u003e(D) \u003c/strong\u003etranswell to evaluate cell migration ability in different groups; \u003cstrong\u003e(E)\u003c/strong\u003e Western Blot analysis of protein expression levels of PCNA, Cyclin B1, Cyclin D1, and β-actin in different groups; \u003cstrong\u003e(F)\u003c/strong\u003eWestern Blot analysis of cell proliferation ability in different groups; \u003cstrong\u003e(G) \u003c/strong\u003eScratch assay to evaluate cell migration ability in different groups; \u003cstrong\u003e(H) \u003c/strong\u003etranswell to evaluate cell migration ability in different groups.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8191897/v1/2961c9ed8f4b34a3da9b756c.jpeg"},{"id":101304094,"identity":"8667ecb4-be43-41e2-9a6b-0f9931adef76","added_by":"auto","created_at":"2026-01-28 10:01:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1270373,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8191897/v1/2d5fde31-d23d-4e6e-9ea6-a1ee0ce8521f.pdf"},{"id":97734041,"identity":"1d1b9b9c-9bd9-4760-808b-651ee2ce2d88","added_by":"auto","created_at":"2025-12-08 18:56:43","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1726076,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8191897/v1/4529a0eadc402db877a09dd8.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Research on the mechanism by which USP18 promotes colorectal cancer progression through stabilizing KRT16 expression","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eColorectal cancer (CRC) is the third leading cause of cancer-related deaths globally[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Early diagnosis and treatment can significantly improve survival rates[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Despite advancements in understanding the etiology, pathogenesis, and epidemiology of CRC, its molecular mechanisms remain incompletely elucidated. Additionally, the increasing incidence of CRC in younger populations and distant metastasis remain major contributors to patient mortality[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, investigating the underlying mechanisms of CRC progression and identifying novel therapeutic targets are crucial for improving patient outcomes.\u003c/p\u003e\u003cp\u003eOur previous studies demonstrated that USP18 promotes colorectal cancer cell proliferation and may serve as a potential diagnostic biomarker[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. USP18, a deubiquitinating enzyme, is upregulated in various cancers and functions to inhibit apoptosis and promote tumorigenesis[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, its downstream regulatory targets in CRC remain unclear.\u003c/p\u003e\u003cp\u003eKeratins, intermediate filament proteins, maintain structural integrity in epithelial cells, regulate cell size, determine organelle positioning, and influence protein translation and cell polarity[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Fifty-four keratins have been identified in human epithelial cells, including 28 type I (acidic forms, K9\u0026ndash;K28) and 26 type II (basic forms, K1\u0026ndash;K8 and K71\u0026ndash;K74) proteins[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Studies suggest that keratins influence tumorigenesis and metastasis. In our in-depth investigation of the downstream regulatory mechanisms of USP18, we have noted that the keratin family member KRT16, due to its aberrant expression in various epithelial-derived tumors and close association with malignant progression, has emerged as a noteworthy potential target. For instance, Elevated KRT16 mRNA expression is associated with reduced recurrence-free survival (RFS) in metastatic breast cancer[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and KRT16 is upregulated in oral squamous cell carcinoma (OSCC) cell lines and tumor tissues[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Understanding the mechanistic role of KRT16 in tumorigenesis and its functional impact is essential for both basic research and therapeutic targeting.\u003c/p\u003e\u003cp\u003eIn this study, we employed mass spectrometry, TCGA database analysis, clinical tumor tissues, and in vitro experiments to investigate the interaction between USP18 and KRT16. We aimed to determine whether KRT16 serves as a biomarker for CRC and whether USP18 regulates KRT16 expression to drive CRC progression. Our findings provide a theoretical foundation for identifying novel therapeutic targets in CRC.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Clinical specimens\u003c/h2\u003e\u003cp\u003eOne hundred colorectal cancer tissue specimens and paired clinical-pathological data were collected from patients treated at Jiaxing First Hospital between January 2019 and December 2020 (67 males, 33 females). All cases were pathologically confirmed, with no prior radiotherapy or chemotherapy. This study was approved by the Ethics Committee of Jiaxing First Hospital (2024-LY-603). Clinical trial number: not applicable.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Cells and reagents\u003c/h2\u003e\u003cp\u003eThe human colon cancer cell line SW480 was purchased from Procell Life Science \u0026amp; Technology Co., Ltd. Fetal bovine serum (40131ES76), reverse transcription kits (11141ES), SYBR Green Master Mix (11184ES), and CCK-8 (40203ES76) were obtained from Yeasen Biotechnology. RPMI-1640 medium (MA0215) was purchased from Meilun Biotechnology. Penicillin-streptomycin solution (C0222), IP lysis buffer (P0013), RIPA buffer (P0013B), and Lipo8000\u0026trade; transfection reagent (C0533) were acquired from Beyotime Biotechnology(Shanghai,China). The Real EnVision Detection System K5007 (DAKO) was used for immunohistochemistry(Copenhagen, Denmark). MG132 (S2619), Protein A/G magnetic beads (B23201), and Anti-Flag magnetic beads (B26101) were purchased from Selleck. Antibodies against USP18 (A16739), KRT16 (A7493), PCNA (A0264), Cyclin B1 (A16038), Cyclin D1 (A11022), and β-actin (AC004) were purchased from ABclonal(Wuhan,China). IRDye 800CW goat anti-rabbit (926-32211) and anti-mouse (926-32210) secondary antibodies were obtained from LI-COR. Trizol reagent (15596026CN) and BCA protein assay kits were purchased from Thermo Fisher Scientific.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Cell culture and transfection\u003c/h2\u003e\u003cp\u003eSW480 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37\u0026deg;C under 5% CO₂.SW480 cells were seeded into 6-well plates 24 hours prior to transfection to achieve 70\u0026ndash;80% confluency. Plasmid DNA was diluted in serum-free RPMI-1640 medium and mixed with Lipo8000\u0026trade; transfection reagent. The mixture was added dropwise to cells and incubated for 48 hours.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Western blotting\u003c/h2\u003e\u003cp\u003eProteins were extracted using RIPA buffer and quantified via BCA assay. Samples (20 \u0026micro;g/lane) were separated by SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk. Membranes were incubated with primary antibodies (USP18: 1:500; KRT16: 1:1000; PCNA: 1:1000; Cyclin B1: 1:1000; Cyclin D1: 1:1000; β-actin: 1:3000) overnight at 4\u0026deg;C. After incubation with IRDye 800CW secondary antibodies (1:10,000), protein bands were visualized using an Odyssey DLx Imaging System (LI-COR) and analyzed with ImageJ.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Co-Immunoprecipitation (Co-IP)\u003c/h2\u003e\u003cp\u003eUpon transfection with USP18, the proteasome inhibitor MG132 (MCE, Cat# HY-13259) was added 6 hours prior to cell collection. After harvesting, total proteins were extracted using IP lysis buffer (Beyotime, Cat# P0013). A small portion of the total protein was reserved as the Input control, while the remaining protein samples were equally divided and incubated overnight at 4\u0026deg;C with rotation with either Protein A/G immunoprecipitation magnetic beads (MCE, Cat# HY-K0202) pre-coated with IgG isotype control antibody (CST, Cat# 3900S) or Anti-Flag magnetic beads (MCE, Cat# HY-K0207). The next day, samples were denatured with 1\u0026times;Loading Buffer (Beyotime, Cat# P0015) at 95\u0026deg;C for 10 minutes, followed by protein interaction analysis via Western Blot.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Immunofluorescence (IF)\u003c/h2\u003e\u003cp\u003eSeeding and culturing adherent cells on sterile glass coverslips until they reach 50\u0026ndash;70% confluence. The cells are then washed with phosphate-buffered saline (PBS) and fixed, typically with 4% paraformaldehyde, to preserve cellular architecture. Following fixation, cells are permeabilized with a detergent solution such as 0.1% Triton X-100 to allow antibody access to intracellular targets. Non-specific binding sites are subsequently blocked using a solution of bovine serum albumin (BSA). The cells are then incubated with a primary antibody specific to the protein of interest, diluted in blocking buffer, after which unbound antibody is removed by washing. A fluorophore-conjugated secondary antibody, directed against the host species of the primary antibody, is applied to visualize the target. Finally, the coverslips are mounted onto microscope slides using an anti-fade mounting medium containing a nuclear counterstain like DAPI, and the samples are visualized using fluorescence.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Immunohistochemistry (IHC)\u003c/h2\u003e\u003cp\u003eTissue sections were deparaffinized in xylene and rehydrated through a graded ethanol series. Antigen retrieval was performed by heating the sections in citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide for 15 minutes. Subsequently, the sections were incubated overnight at 4\u0026deg;C with primary antibodies against USP18 (1:800) and KRT16 (1:400). For the negative control, the primary antibody was replaced with PBS. After primary antibody incubation, the sections were treated with an enhanced enzyme-labeled goat anti-mouse/rabbit IgG polymer (EnVision\u0026trade; system)\u0026mdash;a method that conjugates the secondary antibody and enzyme into a polymer (i.e., EnVision) to amplify the signal. Color development was achieved using a freshly prepared DAB substrate solution. Finally, the sections were counterstained with hematoxylin, dehydrated, and mounted with neutral balsam. IHC results were assessed based on the proportion of positive cells and the staining intensity. The presence of brown-yellow or brown granules in the cytoplasm was considered positive staining. All slides were independently evaluated by two senior pathologists under double-blind conditions. Five random fields per section were examined under a 400\u0026times; light microscope and scored according to the following criteria:percentage of positive cells: 0% (0 points); \u0026le;10% (1 point); 11%\u0026ndash;50% (2 points); 51%\u0026ndash;70% (3 points); \u0026ge;71% (4 points). staining intensity: No staining (0 points); pale yellow (1 point); brown-yellow (2 points); brown (3 points).The final staining score (ranging from 0 to 12) was calculated by multiplying the score for the percentage of positive cells by the staining intensity score. A final score of \u0026ge;\u0026thinsp;6 was defined as high expression, and a score of \u0026lt;\u0026thinsp;6 was defined as low expression.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Wound healing assay\u003c/h2\u003e\u003cp\u003eSW480 cells were transfected for 48 hours, and a scratch was created using a 200 \u0026micro;l pipette tip. Cells were washed with PBS and cultured in serum-free medium. Migration was monitored at 0 and 24 hours using an inverted microscope. Wound closure was quantified with ImageJ.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Transwell migration assay\u003c/h2\u003e\u003cp\u003eCells in the logarithmic growth phase were trypsinized, resuspended in serum-free medium, and adjusted to the appropriate concentration. A 100\u0026ndash;200 \u0026micro;L cell suspension was added to the upper chamber, which was then inserted into a 24-well plate. The plate was incubated at 37\u0026deg;C with 5% CO₂ for 24 h. After incubation, the Transwell chamber was carefully removed, and non-migrated cells on the upper surface of the membrane were gently wiped off using a moistened cotton swab. The chamber was fixed in 4% paraformaldehyde for 15\u0026ndash;30 min, followed by staining with 0.1% crystal violet for 10\u0026ndash;30 min. Excess stain was removed by washing with PBS until no floating color remained, and the membrane was air-dried. Migrated cells on the lower surface of the membrane were counted under a microscope (100\u0026times; or 200\u0026times; magnification) by randomly selecting 5\u0026ndash;12 fields of view.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Cell Counting Kit-8 (CCK-8) assay\u003c/h2\u003e\u003cp\u003eTransfected SW480 cells (2,000 cells/well) were seeded into 96-well plates. Cell viability was assessed at 24, 48, 72, and 96 hours using CCK-8 reagent. Absorbance at 450 nm was measured.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Quantitative Real-time Polymerase Chain Reaction (qPCR) analysis\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted with Trizol, reverse-transcribed into cDNA, and amplified using SYBR Green Master Mix. KRT16 expression was normalized to β-Actin (ACTB) and calculated via the 2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔCt method. Primer sequences:\u003c/p\u003e\u003cp\u003eHomo KRT16:\u003c/p\u003e\u003cp\u003eForward: 5\u0026prime;-AAAGGAACCGCCCCAAATCT-3\u0026prime;\u003c/p\u003e\u003cp\u003eReverse: 5\u0026prime;-GTATCTCTGTGCCCGGACTG-3\u0026prime;\u003c/p\u003e\u003cp\u003eHomo ACTB:\u003c/p\u003e\u003cp\u003eForward: 5\u0026prime;-GAGAAAATCTGGCACCACACC-3\u0026prime;\u003c/p\u003e\u003cp\u003eReverse: 5\u0026prime;-GGATAGCACAGCCTGGATAGCAA-3\u0026prime;\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12 Statistical Analysis\u003c/h2\u003e\u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Comparisons between groups were analyzed using Student\u0026rsquo;s t-test (normally distributed data) or Wilcoxon rank-sum test (non-normal data). Spearman\u0026rsquo;s correlation and log-rank tests were used for survival analysis. All analyses were performed using GraphPad Prism 10.0 and Xiantao Academic (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.xiantaozi.com/\u003c/span\u003e\u003cspan address=\"https://www.xiantaozi.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.1 KRT16 is a potential downstream target of USP18\u003c/h2\u003e\u003cp\u003eBased on our mass spectrometry analysis of USP18-interacting proteins, we identified keratin 16 (KRT16) as a strong candidate binding partner (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To explore the functional consequence of this interaction, we first established a robust USP18 overexpression system, which resulted in a dramatic increase in USP18 mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Notably, this forced expression of USP18 led to a significant upregulation of KRT16 protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), suggesting a potential regulatory relationship. We further confirmed the physical association between these two proteins by demonstrating their direct interaction under physiological conditions using an endogenous co-immunoprecipitation assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Given the known deubiquitinating function of USP18, we hypothesized that it might stabilize KRT16 by modulating its ubiquitination. Consistent with this, overexpression of USP18 substantially reduced the ubiquitination level of KRT16 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Conversely, knockdown of USP18 effectively enhanced KRT16 ubiquitination (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), providing compelling evidence that USP18 negatively regulates KRT16's ubiquitin-mediated degradation. Finally, immunofluorescence microscopy visually confirmed the functional interaction between USP18 and KRT16, revealing their distinct colocalization within cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.2 KRT16 expression and prognostic significance in CRC\u003c/h2\u003e\u003cp\u003eBased on the comprehensive bioinformatics analysis of the TCGA database, we observed a significant overexpression of KRT16 in colorectal cancer (CRC) tissues relative to normal counterparts (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). A positive correlation was further identified between the expression levels of KRT16 and USP18 (\u003cem\u003eR\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.308, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The diagnostic potential of KRT16 for CRC was underscored by receiver operating characteristic (ROC) curve analysis, which yielded a high area under the curve(AUC) of 0.899 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). From a prognostic standpoint, Kaplan\u0026ndash;Meier survival analysis revealed that elevated KRT16 expression was associated with markedly shorter overall survival, corresponding to a 1.5-fold increase in mortality risk (Hazard Ratio [HR]\u0026thinsp;=\u0026thinsp;1.55, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.029, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). These findings were further corroborated by our validation cohort. Western blot analysis of 14 paired clinical samples from Jiaxing First Hospital confirmed a significant upregulation of KRT16 protein in CRC tissues compared to adjacent normal mucosa (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Consistent with this, immunohistochemical staining of a larger series of 100 paraffin-embedded CRC specimens demonstrated significantly higher protein levels of both KRT16 and USP18 in cancerous tissues (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Furthermore, clinicopathological analysis indicated that high KRT16 expression was significantly associated with advanced T stage (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008) and showed a strong positive correlation with USP18 expression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), reinforcing its clinical relevance in CRC progression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eExpression of KRT16 in 100 colorectal cancer tissues and its relationship with clinicopathological characteristics [n (%)]\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\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\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCharacteristics\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eTotal (n\u0026thinsp;=\u0026thinsp;100)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eKRT16\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eχ\u0026sup2;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLow\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGender, n(%)\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\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eχ\u0026sup2;=0.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.353\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\u003e67 (67.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e24 (61.54)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e43 (70.49)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\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\u003e33 (33.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e15 (38.46)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e18 (29.51)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAges, n(%)\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\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eχ\u0026sup2;=0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.657\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\u003e36 (36.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e13 (33.33)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e23 (37.70)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\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\u003e64 (64.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e26 (66.67)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e38 (62.30)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLocation, n(%)\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\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eχ\u0026sup2;=1.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.210\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eColon\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e59 (59.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20 (51.28)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e39 (63.93)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRectum\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e41 (41.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e19 (48.72)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e22 (36.07)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDiameter, n(%)\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\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eχ\u0026sup2;=0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.828\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;5cm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e73 (73.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e28 (71.79)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e45 (73.77)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;5cm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e27 (27.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e11 (28.21)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16 (26.23)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTumor type, n(%)\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\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eχ\u0026sup2;=0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOther\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3 (3.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1 (2.56)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2 (3.28)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAdenocarcinoma\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e97 (97.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e38 (97.44)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e59 (96.72)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDifferentiation grade, n(%)\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\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.076\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5 (5.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1 (2.56)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4 (6.56)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3 (3.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3 (7.69)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0 (0.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eModerate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e92 (92.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e35 (89.74)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e57 (93.44)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT stage, n(%)\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\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eχ\u0026sup2;=7.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.008\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT1\u0026thinsp;~\u0026thinsp;T2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e18 (18.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e12 (30.77)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6 (9.84)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT3\u0026thinsp;~\u0026thinsp;T4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e82 (82.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e27 (69.23)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e55 (90.16)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN stage, n(%)\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\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eχ\u0026sup2;=1.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.277\u003c/p\u003e\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\u003e60 (60.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e26 (66.67)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e34 (55.74)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN1\u0026thinsp;~\u0026thinsp;N2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e40 (40.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e13 (33.33)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e27 (44.26)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eM stage, n(%)\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\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eχ\u0026sup2;=0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.000\u003c/p\u003e\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\u003e96 (96.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e37 (94.87)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e59 (96.72)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\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\u003e4 (4.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2 (5.13)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2 (3.28)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePathological stage, n(%)\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\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eχ\u0026sup2;=1.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.277\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eⅠ~Ⅱ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e60 (60.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e26 (66.67)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e34 (55.74)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eⅢ~Ⅳ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e40 (40.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e13 (33.33)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e27 (44.26)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUSP18, n(%)\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\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eχ\u0026sup2;=35.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e36 (36.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e28 (71.79)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8 (13.11)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e64 (64.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e11 (28.21)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e53 (86.89)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.3 KRT16 knockdown attenuates USP18-driven CRC proliferation and migration\u003c/h2\u003e\u003cp\u003eTo investigate the functional role of KRT16, we transfected CRC cells with sh-KRT16 plasmids. qPCR confirmed efficient knockdown (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Western blot analysis of proliferation markers (PCNA, Cyclin B1, and Cyclin D1) revealed that KRT16 silencing significantly suppressed their protein expression levels compared to the control group (all P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Scratch wound healing and Transwell assays further demonstrated that KRT16 knockdown inhibited the migratory capacity of colon cancer cells (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). Subsequently, cells were co-transfected with sh-KRT16 and USP18 overexpression plasmids. Western blot analysis showed that while KRT16 interference reduced the expression of proliferation markers, USP18 overexpression enhanced their expression. Notably, in the co-transfection group, the USP18-induced upregulation of these markers was significantly attenuated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). CCK-8 assays corroborated these findings: KRT16 knockdown suppressed cell proliferation, USP18 overexpression promoted it, and the pro-proliferative effect of USP18 was partially reversed in co-transfected cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). These results suggest that USP18 likely regulates proliferative capacity through KRT16. Similarly, scratch and Transwell assays confirmed that KRT16 knockdown impaired cell migration, USP18 overexpression enhanced it, and co-transfection counteracted the promigratory effect of USP18 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG,H). Collectively, these data indicate that USP18 promotes colon cancer cell proliferation and migration by modulating KRT16.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eColorectal cancer (CRC) is a common malignant tumor of the digestive tract and the third leading cause of cancer-related deaths. Due to its insidious clinical manifestations, CRC is often diagnosed at advanced stages, with high rates of postoperative recurrence and metastasis, resulting in poor prognosis and low 5-year survival rates[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Although treatment options for CRC have expanded in recent years, including surgery, radiotherapy, chemotherapy, and immunotherapy, the heterogeneity and metastatic potential of CRC continue to pose significant challenges. Consequently, the 5-year survival rate for advanced CRC patients remains unsatisfactory. Therefore, elucidating the molecular mechanisms underlying CRC progression and identifying novel biomarkers are critical for improving early diagnosis, prognosis prediction, and targeted therapy.\u003c/p\u003e\u003cp\u003eOur previous study demonstrated that elevated USP18 expression is associated with CRC proliferation and metastasis[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, the precise mechanisms by which USP18 promotes CRC remain unclear. As a deubiquitinating enzyme, USP18 likely exerts its oncogenic effects by stabilizing key regulatory proteins. In this study, we employed co-immunoprecipitation (Co-IP) combined with mass spectrometry to identify potential downstream targets of USP18, ultimately identifying keratin 16 (KRT16) as a candidate.\u003c/p\u003e\u003cp\u003eKeratins are well-established tumor markers in clinical practice, implicated in epithelial metaplasia, malignant transformation, and metastasis. Based on their isoelectric points, keratins are classified into acidic type I (CK9\u0026ndash;CK20) and basic type II (CK1\u0026ndash;CK8) subtypes[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Previous studies have shown that CK8, CK18, and CK19 are overexpressed in CRC tissues compared to adjacent normal tissues and may contribute to CRC progression[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. For instance, CK18 upregulation activates the PI3K-AKT pathway and epithelial-mesenchymal transition (EMT), thereby promoting CRC cell proliferation, invasion, and metastasis[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The KRT16 gene, located on chromosome 17q21.23, has been implicated in the pathogenesis of various cancers, including nasopharyngeal carcinoma, breast cancer, and lung adenocarcinoma, where it exhibits oncogenic properties and correlates with poor prognosis[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, the role of KRT16 in CRC remains largely unexplored.\u003c/p\u003e\u003cp\u003eIn this study, we found that KRT16 expression was significantly higher in CRC tissues than in adjacent normal tissues, suggesting its potential diagnostic value. Furthermore, KRT16 protein expression and T stage were independent prognostic factors for CRC patients, with high KRT16 expression associated with shorter overall survival. These findings indicate that KRT16 may play a pivotal role in CRC progression and warrant further clinical validation.\u003c/p\u003e\u003cp\u003ePrevious studies have reported that KRT16 is overexpressed in lung adenocarcinoma cells, and its knockdown suppresses proliferation, invasion, migration, and EMT[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In this study, we used shRNA-mediated KRT16 knockdown and demonstrated that silencing KRT16 significantly inhibited the proliferative and migratory capacities of CRC cells. Given the positive correlation and direct interaction between USP18 and KRT16 in CRC, we co-transfected USP18 overexpression plasmids with KRT16 shRNA into CRC cells. The results showed that KRT16 knockdown attenuated the tumor-promoting effects of USP18, suggesting that USP18 may drive CRC progression, at least in part, by stabilizing KRT16.\u003c/p\u003e\u003cp\u003eIn summary, KRT16 is overexpressed in CRC and serves as an independent prognostic marker for poor survival. The USP18-KRT16 axis may represent a promising therapeutic target for CRC intervention. Further studies are needed to validate these findings in larger clinical cohorts and explore the detailed molecular mechanisms underlying USP18-mediated KRT16 stabilization.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.Z. was responsible for study implementation and data collection, and wrote the manuscript. X.C. and X.L. conducted data analysis and statistics. H.Y. and Z.G. provided technical and material support. W.W. supervised the study, reviewed the manuscript, and acquired funding. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZhejiang Provincial Medical and Health Science and Technology Project (2025KY1584); Jiaxing Key Medical Discipline (2023-ZC-017); Jiaxing Civil Science and Technology Innovation Special Project (2020AY30008)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe procedures used in this study adhere to the tenets of the Declaration of Helsinki. And all experimental procedures were approved by the Ethics Committee of Jiaxing First Hospital (2024-LY-603).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from each participant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSung H., Ferlay J., Siegel R.L., Laversanne M., Soerjomataram I., Jemal A. and Bray F. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 2021;71(3): 209-249. DOI:10.3322/caac.21660.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eStark U.A., Frese T., Unverzagt S., Danner D., Unverzagt M. and Herrmann K. What is the effectiveness of various invitation methods to colonoscopy in early detection of colorectal cancer? Protocol of a systematic review. Syst. Rev. 2020;9(1):49. DOI:10.1186/s13643-020-01312-x.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMolecular Diagnostics Group of Chinese Society of Laboratory Medicine. Chinese expert consensus on laboratory diagnostic techniques for early colorectal cancer and precancerous lesions. Chin. J. Lab. Med.\u0026nbsp;2021;44(5): 372-380.\u003c/li\u003e\n \u003cli\u003eZhang L., Zhang N., Li X., Yang H., Lu L. and Wu W. High expression of USP18 is associated with the growth of colorectal carcinoma. Histol. Histopathol.2021;36(6): 697-704. DOI:10.14670/HH-18-346.\u003c/li\u003e\n \u003cli\u003eGeng L., Liu F., Yang L., Wang Y., Wang X. and Zhang Y. USP18 promotes proliferation, invasion, and migration of head and neck squamous cell carcinoma by deubiquitinating PLK1. Exp. Cell Res. 2024;114284. DOI:10.1016/j.yexcr.2024.114284.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMiyauchi S., Arimoto K.I., Liu M., Xu J., Zhang X., Zhou Y., Xu G., Li S., Wei S., Zhou J., Zhang D.E. and Zhang X. Reprogramming of tumor-associated macrophages via Nedd4-mediated CSF1R degradation by targeting USP18. Cell Rep. 2023;42(12):113560. DOI:10.1016/j.celrep.2023.113560.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eDmello C., Srivastava S.S., Tiwari R., Chaudhari P.R., Zhang L., Singh S., Bayat A., Ganguly K., Dmello Y. and Singh K.K. Multifaceted role of keratins in epithelial cell differentiation and transformation. J. Biosci. 2019;44(2).\u003c/li\u003e\n \u003cli\u003eTaikowski K., Rudinsky A.J., Louke D.S., Brisbin J., Weng H.Y. and Selmic L.E. Plasma cytokeratin 18 and fecal alpha-1 antitrypsin concentrations in dogs with osteosarcoma receiving carboplatin chemotherapy. Vet. Med. Sci. 2021;7(2):385-392. DOI:10.1002/vms3.392.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSi H., Liu R., Xiong Z.Y., Li J., Liu S. and Wang X. Correlation between cytokeratin expression and clinicopathological features in colorectal cancer. J. Capital Med. Univ. 2022;43(1): 127-131.\u003c/li\u003e\n \u003cli\u003eShao N., Yuan K., Zhang Y., Zhao X., Zhang W. and Wang X. Identification of key candidate genes and pathways in ER-negative/HER2-negative breast cancer by bioinformatics analysis. J. BUON. 2018;23(4): 891-901.\u003c/li\u003e\n \u003cli\u003eKengkarn S., Petmitr S., Boonyuen U., Reamtong O., Kalpongnukul N. and Weeraphan C. Identification of novel candidate biomarkers for oral squamous cell carcinoma via whole gene expression profiling. Pathol. Oncol. Res. 2020;26(4):2315-2325.DOI:10.1007/s12253-020-00828-w.\u003c/li\u003e\n \u003cli\u003eHuang L.B., Huang Q.S. and Yang L. Epidemiology and prevention of colorectal cancer globally and in China: Interpretation of \u0026quot;Global Cancer Statistics 2022\u0026quot;. Chin. J. Bases Clin. General Surg. 2024;31(5): 530-537.\u003c/li\u003e\n \u003cli\u003eJacob J.T., Coulombe P.A., Kwan R. and Omary M.B. Types I and II keratin intermediate filaments. Cold Spring Harb. Perspect. Biol. 2018;10(4). DOI:10.1101/cshperspect.a018275.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHinz S., Hendricks A., Wittig A., Schafmayer C., Tepel J., Kalthoff H., Becker T. and R\u0026ouml;der C. Detection of circulating tumor cells with CK20 RT-PCR is an independent negative prognostic marker in colon cancer patients: A prospective study. BMC Cancer.2017;17(1):53. DOI:10.1186/s12885-016-3035-1.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMoll R., Divo M. and Langbein L. The human keratins: biology and pathology. Histochem. Cell Biol. 2008; 129(6): 705-733. DOI:10.1007/s00418-008-0435-6.\u003c/li\u003e\n \u003cli\u003eKurtovic M., Pitesa N., Conkas J., Krajina R., Spagnoli G., Jukic I., Situm M., Jonjic N. and Kovacevic D. GLI transcriptional targets S100A7 and KRT16 show upregulated expression in epidermis overlying melanoma tumors. Int. J. Mol. Sci. 2024;25(11). DOI:10.3390/ijms25116084.\u003c/li\u003e\n \u003cli\u003eWang W., Zhu L., Zhou J., Li Y., Li Y., Mao X., Zhang X. and Xu Y. (2023). Targeting the KRT16-vimentin axis for metastasis in lung cancer. Pharmacol. Res. 2023;193: 106818. DOI:10.1016/j.phrs.2023.106818.\u003c/li\u003e\n \u003cli\u003eYuanhua L., Pudong Q., Wei Z., Ting L., Yan Z., Lin L., Fan Y., Li W., Xiaoyan X. and Tao W. TFAP2A-induced KRT16 acts as an oncogene in lung adenocarcinoma via EMT. Int. J. Biol. Sci. 2019;15(7):1419-1428. DOI:10.7150/ijbs.34076.\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":"Colorectal cancer, USP18, KRT16, Proliferation, Migration","lastPublishedDoi":"10.21203/rs.3.rs-8191897/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8191897/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground This study aims to\u003c/h2\u003e\u003cp\u003einvestigate the effects of Ubiquitin-specific peptidase 18 (USP18) and Keratin16 (KRT16) on the progression of colorectal cancer and their possible mechanism of action.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eThe downstream target protein of USP18 was identified using Co-immunoprecipitation(Co-IP) and mass spectrometry. The clinical significance of KRT16 was validated via bioinformatics analysis of the TCGA database. Immunohistochemical staining detected USP18 and KRT16 expression in CRC tissues, and their relationship with clinicopathological features was analyzed. In vitro functional assays, including Western Blot, scratch tests, and CCK-8 assays, were performed using USP18 overexpression and KRT16 interference plasmids.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eKRT16 was identified as a downstream target of USP18, and USP18 overexpression was shown to deubiquitinate KRT16. Bioinformatics analysis revealed that KRT16 was highly expressed in CRC (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), exhibited significant prognostic value (log-rank P\u0026thinsp;=\u0026thinsp;0.029) and high diagnostic accuracy (AUC\u0026thinsp;=\u0026thinsp;0.899). A positive correlation was found between KRT16 and USP18 expression (R\u0026thinsp;=\u0026thinsp;0.308, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Immunohistochemistry confirmed that both KRT16 and USP18 were significantly upregulated in CRC tissues, with KRT16 expression correlating with T stage (P\u0026thinsp;=\u0026thinsp;0.008). In vitro, KRT16 knockdown inhibited CRC cell proliferation and migration. Importantly, USP18 overexpression enhanced proliferation and migration, which was rescued by KRT16 knockdown.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eKRT16 can be used as one of the markers of colorectal cancer, and USP18 may be involved in the occurrence and development of colorectal cancer by regulating the expression of KRT16.\u003c/p\u003e","manuscriptTitle":"Research on the mechanism by which USP18 promotes colorectal cancer progression through stabilizing KRT16 expression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-08 18:56:38","doi":"10.21203/rs.3.rs-8191897/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":"f90fa3d7-4dfe-49e3-a262-b7b238e66f14","owner":[],"postedDate":"December 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-28T09:52:43+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-08 18:56:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8191897","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8191897","identity":"rs-8191897","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}