The Molecular Mechanism Of UTP18 In The Colorectal Adenoma-Carcinoma Sequence

Background:Colorectal cancer(CRC)is a common malignant tumor of the digestive tract.With changes in dietary structure and an aging population,the incidence of CRC continues to rise,posing a serious threat to public health.Colorectal adenoma is the main precursor lesion of CRC,and about 85%of CRC cases evolve from adenomas.Early detection and treatment of colorectal adenomas can help reduce the incidence of CRC.Since most patients with colorectal adenomas have no obvious clinical symptoms,early detection of colorectal adenomas requires colonoscopy screening,and colonoscopic resection is the preferred treatment for colorectal adenomas.However,colonoscopy is an invasive examination with certain limitations.Patients with severe rectal stenosis or acute severe colitis are not suitable for the examination,and complications such as intestinal bleeding and perforation may occur during the examination.In addition,colorectal adenomas are prone to recurrence after resection,and patients need regular colonoscopy monitoring.However,the benefits of regular monitoring are still controversial,and there is still a lack of clinical biomarkers for the effective diagnosis of colorectal adenomas.Therefore,studying the molecular mechanisms of colorectal adenoma carcinogenesis and identifying driving factors are of great significance for effective monitoring and prevention of carcinogenesis.Methods:1.Proteomics and bioinformatics screening of potential driving factors for colorectal adenoma carcinogenesis and exploration of clinical value(1)TMT quantitative proteomics and bioinformatics analysis were performed on normal intestinal epithelial tissues,adenoma tissues,and cancer tissues from 7 patients with CRC combined with adenoma,to screen potential driving factors for colorectal adenoma carcinogenesis.(2)ROC curve and survival analysis were performed on tumor databases and experimental data to explore the potential clinical value of UTP18.2.In vitro and in vivo studies of the function of UTP18 in colorectal adenoma carcinogenesis(1)Human and mouse intestinal adenoma organoids were constructed,and lentivirus transfection technology was used to knock down or overexpress UTP18 on adenoma organoids to detect the effect of UTP18 on cell viability and apoptosis in adenoma organoids.(2)CCK8,colony formation assay,transwell,and nude mouse tumor formation experiments were used to study the effect of UTP18 on the malignant degree of CRC cells.3.Molecular mechanisms of UTP18 in regulating the proliferation of colorectal adenoma organoids and CRC cells(1)Transcriptomic analysis was performed on colorectal adenoma organoids overexpressing UTP18,and proteomic analysis was performed on CRC cell lines with UTP18 knockdown.Multi-omics analysis and bioinformatics were used to explore the main signaling pathways and downstream regulatory factors regulated by UTP18.(2)Nuclear function of UTP18:Polysome profiling was used to detect the effect of UTP18 on ribosome biogenesis.(3)Extranuclear function of UTP18:Nuclear-cytoplasmic fractionation,immunoprecipitation,and bioinformatics were used to study the types and sites of post-translational modifications that regulate UTP18 nucleocytoplasmic transport,and to detect the effect of UTP18 nucleocytoplasmic transport on the proliferation of CRC cells.RNA-binding protein immunoprecipitation and mRNA decay assays were used to examine the regulatory role of UTP18 on p21 mRNA.(4)Molecular docking and IC50 experiments were performed to validate the feasibility of UTP18 as a potential therapeutic target for CRC.4.Evaluating the clinical application value of UTP18 in the diagnosis and prognostic assessment of CRC through pathological and serological studies(1)Immunohistochemistry experiments were performed to detect the expression of UTP18 in the first resected adenoma tissues of 30 non-recurrent colorectal adenoma patients and 40 recurrent colorectal adenoma patients.The clinical potential of UTP18 in predicting colorectal adenoma recurrence was evaluated.(2)ELISA was used to measure the level of UTP18 in the serum of 230 CRC patients,12 ulcerative colitis patients,18 gastric cancer patients,and 143 healthy controls,to evaluate the clinical significance of UTP18 in the diagnosis and prognostic assessment of CRC.Results:1.Screening and preliminary exploration of potential driver factors for colorectal adenoma carcinogenesis and clinical value(1)Proteins consistently upregulated from normal colonic epithelium to adenoma to cancer tissues were mainly enriched in the ribosome biogenesis signaling pathway,among which UTP18 was one of the key factors in the protein interaction network of this pathway.(2)UTP 18 expression increased during the colorectal adenoma-carcinoma sequence and showed potential to distinguish normal colonic epithelium,adenoma,and cancer tissues,which was associated with poor prognosis in patients.2.The function and characteristics of UTP18 in colorectal adenoma carcinogenesis(1)Overexpression of UTP 18 promotes cell viability of human and mouse colorectal adenoma organoids increased the expression of cell proliferation markers Ki67 and PCNA,while knocking down UTP 18 induced apoptosis of adenoma organoids.(2)Knocking down UTP 18 inhibited the proliferation,invasion,and migration ability of CRC cell lines HCT116 and SW480,and reduced the tumor formation ability of CRC cells in nude mice.(3)Overexpression of UTP 18 in normal intestinal epithelial cell line NCM460 and CRC cell line HCT116 had no significant effect on cell proliferation.3.Molecular mechanisms of UTP18 regulating the colorectal adenoma-carcinoma transformation through multiple pathways(1)UTP 18 exerted its effects by regulating the cell cycle signaling pathway.Knockdown of UTP18 results in cell cycle arrested at the G0/G1 phase in SW480 and HCT116 cells.(2)Overexpression of UTP 18 promoted ribosome biogenesis in adenoma organoids.However,UTP 18 had differential effects on ribosome biogenesis in different CRC cell types.Knockdown of UTP 18 significantly reduced the synthesis of the 40S subunit in SW480 cells,while having no significant effect on HCT116 cells.Overexpression of UTP 18 inhibited ribosome biogenesis in SW480 cells,and had no significant effect on ribosome biogenesis in HCT116 cells.(3)The nucleocytoplasmic transport of UTP 18 increased during colorectal adenoma-carcinoma transformation.UTP 18 nucleocytoplasmic transport was regulated by SUMOylation,with SUMO1 modification at K10 and K183 predicted as the SUMOylation sites.The nucleocytoplasmic transport of UTP 18 increased the proliferation ability of CRC cells significantly.(4)Analysis of the GEO database showed a negative correlation between UTP 18 and p21 mRNA expression.Nucleocytoplasmic transport of UTP 18 increased its binding to p21 mRNA,reducing the stability of p21 mRNA.(5)Small molecule inhibitors OICR-9429 and WDR5-IN-4 bound to the WD domain of UTP18.After overexpression of UTP18,the IC50 of OICR-9429 and WDR5-IN-4 on CRC cells increased,indicating that UTP18 is a potential therapeutic target for CRC.4.Evaluation of the potential clinical significance of UTP18 in CRC diagnosis and prognosis(1)The expression of UTP18 in the first excised adenoma tissues of colorectal adenoma recurrence patients were significantly higher than that of non-recurrence patients,indicating that UTP18 has the potential to predict adenoma recurrence.(2)The expression of UTP18 in the serum of CRC patients was significantly higher than that of healthy controls.Combined with CEA detection,the diagnostic efficiency of CRC was significantly improved.High expression of UTP18 in serum was associated with poor prognosis of CRC.Conclusion:UTP18 was gradually upregulated during the process of colorectal adenoma carcinogenesis and was a potential biomarker for distinguishing between normal,adenoma,and cancer tissues in the colon,as well as predicting prognosis.Overexpression of UTP18 increased the activity of colorectal adenoma organoids,while knockdown of UTP18 induced apoptosis of colorectal adenoma organoids,reduced the malignancy of CRC cells,and caused cell cycle arrest in the G0/G1 phase.In terms of mechanisms,UTP18 regulated colorectal adenoma carcinogenesis through multiple pathways.Overexpression of UTP18 promoted the biogenesis of colorectal adenoma ribosomes,while knockdown of UTP18 inhibitd the synthesis of 40S subunits in microsatellite stable CRC cells.In addition,SUMOylation modification regulated UTP18 nucleocytoplasmic translocation,and UTP18 promoted colorectal adenoma carcinogenesis by mediating the stability of p21 mRNA.In terms of clinical significance,UTP18 had potential clinical significance in predicting colorectal adenoma recurrence and serological diagnosis of CRC.