14,15-Epoxyeicosatrienoic Acids Promotes Intestinal Adenoma Growth And Upregulates Ribosomal Proteins Expression

Background and Objective:Colorectal cancer（CRC）is one of the common malignant tumors seriously harmful to human health.There are multiple factors,stages and steps in the initiation and progression of CRC.Adenomatous polyposis coli（APC）gene is an important tumor suppressor gene in CRC,its inactivation can be seen in 85%of CRC patients in early stages of disease.Many studies have confirmed that lipid metabolites,immune cells,inflammatory cells,cytokines and chemokines are constantly changes in tumor microenvironment.Tumor cells are usually crosstalk with such factors and further promote their development.As an important endogenous lipid signaling molecules,lipid metabolites are also significant in tumorigenesis and progression,thus become hotspots in current study.However,which lipid metabolites changes in the early stage of intestinal tumor and how they affect tumorigenesis and progression still unclear.In addition,except for involved in protein synthesis,ribosomal proteins（RPs）also play role in tumorigenesis and progression,including cell proliferation,apoptosis,migration and invasion.The mutation or overexpression of RPs related genes in tumors is closely related to cell activities,invasion and other malignant phenotype.Rps expression is regulated by many factors,but whether lipid metabolites affect their expression still unclear.In this study,we try to investigate the changes of serum lipid metabolites in the early stage of CRC,how they work,and whether they affect the expression of Rps.Methods:（1）To investigate the changes of serum lipid metabolites in colorectal adenoma mice,serum from C57BL/6 and Apc^Min/+mice in the age of 7week and 24 week was collected respectively.Then,the extensively targeted lipidomics detection was performed by UPLC-MS/MS.（2）In clinical:the expression of Cyp2j2 in colorectal adenocarcinoma and its relationship with pathological stage and overall survival rate was analyzed by clinical tumor database（GEPIA2）.To detect the changes of14,15-EET in serum of CRC patients,serum from healthy volunteers and CRC patients were collected respectively,then the concentration of14,15-EET in serum were detected by ELISA assay.（3）In vivo:to investigate the effect of 14,15-EET（which elevated in serum）on the progression of intestinal adenoma,14,15-EET were intraperitoneally injected into Apc^Min/+mice.Then,methylene blue staining was used to observe the number of intestinal adenomas,H&E staining was used to observe its pathological changes,and Ki67 staining was used to evaluate its proliferation.（4）In vitro:CT26 cells were treated with a certain concentration of14,15-EET.Then,the proliferation of CT26 cells were examined by CCK-8 assay and colony formation assay.The migration and invasion ability of CT26 cells were examined by wound healing assay and transwell assay.（5）To study whether 14,15-EET affected PI3K/AKT and MAPK/ERK signaling pathways,CT26 cells were treated with a different concentration of 14,15-EET,then the phosphorylation levels of AKT and ERK1/2 were detected by Western blot（WB）.To study whether14,15-EET affected epithelial-mesenchymal transition（EMT）process,the expression of E-cadherin,N-cadherin and Vimentin protein were detected by WB.（6）To explore the potential molecular mechanism of 14,15-EET promoting CT26 cells growth,transcriptome analysis of RNA sequencing（RNA-seq）was performed using Illumina high-throughput platform.The potential molecular mechanism was explored by differential gene expression analysis and gene ontology（GO）functional enrichment analysis.The potential molecular were further verified by q PCR in vivo and in vitro at m RNA level.Real-time PCR were used to further verify the potential molecular at m RNA level in vivo and in vitro.Clinical database GEPIA2 and The Human Protein Atlas were used to verify the potential molecular at protein level.Results:（1）14,15-EET is increased in serum of Apc^Min/+mice and it mainly origin from intestinal tissue.（2）14,15-EET is increased in serum of CRC patients and its relate gene Cyp2j2 is overexpression in CRC patients.（3）14,15-EET promotes the pathological process of intestinal adenoma in Apc^Min/+mice and aggravates its malignant degree.（4）14,15-EET promotes the proliferation,migration and invasion of CT26 cells.（5）14,15-EET activates p-AKT and p-ERK1/2 in CT26 cells.14,15-EET also decreased E-cadherin expression,increased N-cadherin and Vimentin expression in CT26 cells.（6）14,15-EET upregulates the expression of ribosomal proteins in CT26cells.Conclusion:（1）14,15-EET is increased in serum of intestinal adenoma mice and colorectal cancer patients,which is a potential biomarker for early diagnosis of intestinal tumor.（2）14,15-EET promotes intestinal adenoma growh and upregulates ribosomal protein genes expression.