Pathogenesis Of Colon Adenocarcinoma And Clinical Value And Mechanism Of Chemokine CXC Subfamily Gene In Colon Adenocarcinoma

This study is divided into four parts:1.Bio informatics analysis of differential genes and prognosis-related gene of colon adenocarcinoma based on TCGA database;2.Diagnosis and prognostic value analysis of chemokine CXC subfamily gene expression in colon cancer patients based on TCGA database;3.The diagnosis and prognostic value of CXCL8,CXCL3 and CXCL1 expression in colonic adenocarcinoma in the First Affiliated Hospital of Guangxi Medical University cohort;4.Using the siRNA interference technique to analyze the biological function of CXCL8 in SW620 and RKO colon adenocarcinoma cell lines.PART 1:BIOINFORMATICS ANALYSIS OF DIFFERENTIAL EXPRESSED GENE S AND PROGNOSIS-RELATED GENE OF COLON ADENOCARCINOMA BASED ON TCGA DATABASEObjective:To identify the genetic characteristics of COAD during its development and to evaluate its potential as a tumor biomarker based on the genome-wide expression microarray dataset of colon adenocarcinoma in the TCGA database.Materials and methods:COAD-related mRNA sequencing data sets were downloaded from the TCGA database.The differentially expressed genes of COAD carcinoma and adjacent noamal tissues were screened by using the R platform's edgeR and DESeq software package,and the intersection was filtered by Venn.Then the R-platform survival software package was used to calculate the genes related to COAD prognosis.We used Venn diagram to filters out differential expressed genes associated with prognosis,and then performed bioinformatics analysis of gene functional enrichment analysis,signal pathway enrichment analysis,and construction of interprotein and intergenic interaction networks.Four important genes were used for diagnostic and prognostic value analysis.Results:A total of 1534 overlapping differentially expressed genes were identified from 480 COAD cancer tissues and 41 adjacent tissues,including 614 up-regulated DEGs and 920 down-regulated DEGs.After adjusting the patient's TNM staging,the R platform Survival package calculated 1111 genes associated with the prognosis of COAD patients,and used the Venn diagram to filter out 107 differential genes associated with COAD prognosis.Bioinformatics analysis enriched the ion transmembrane metabolic pathways such as ion transport,cell differentiation,cell adhesion and other biological functions and Cytokine-cytokine receptor interaction pathway.We performed diagnostic and prognostic value analysis on 42 genes with different expression or prognosis related to CA10,SNAP25,SYT4,and found that POU5F1B,ZBTB7C,ERFE,SCN3A,FJX1,AQP8,GUCA2A,VSTM2A and MMP3 are not only related to prognosis.And has a higher diagnostic value.Conclusion:We explored the molecular mechanisms involved in the development of COAD from the transcriptional level,and analyzed some potential diagnostic and prognostic biomarkers in COAD.PART 2:DIAGNOSIS AND PROGNOSTIC VALUE OF CHEMOKINE CXC SUBFAMILY GENE EXPRESSION IN COLON ADENOCARCINOMA PATIENTS BASED ON TCGA DATABASEObjective:To identify the role of chemokine CXC subfamily gene expression microarray in colon cancer adenocarcinoma(COAD)in the Cancer Genome Atlas(TCGA)database,and to identify its role in the development of COAD and evaluate its potential as a tumor biomarker..Materials and methods:The mRNA expression profiles of chemokine CXC subfamily in COAD in TCGA database and corresponding clinical data were collected.The expression of chemokine CXC subfamily gene in COAD patients'cancer tissues was compared with adjacent normal colontissues to determine the differential expressed genes.The chemokine CXC subfamily gene was analyzed for the diagnosis and prognostic value of COAD.We analyzed the biological function,metabolic pathway,interaction between expressed proteins and gene-gene interaction of chemokine CXC subfamily gene to study the mechanism of chemokine CXC subfamily gene,and identify chemokine CXC subfamily gene that has the greatest influence on the diagnostic value and prognosis of COAD,We performed Gene set enrichment analysis and used the Pearson correlation coefficient to screen the gene co-expressing the gene of CXCL8 in COAD,constructed the co-expression network,to explore the mechanisms by which this gene is involved in the development and prognosis of COAD.Results:Analysis and diagnosis of ROC curves by chemokine CXC subfamily gene expression in 480 COAD tumor tissues and 41 adjacent normal tissues showed that CXCL1,CXCL3,CXCL8 and CXCL12 have a higher diagnosis value of colon cancer.Kaplan-Meier survival curve analysis showed that CXCL1,CXCL3,and CXCL8 were associated with prognosis in COAD patients.Multivariate analysis showed that CXCL8 gene was significantly associated with prognosis in patients with COAD(Adjusted P=0.009,Adjusted HR(95%CI)=0.567(0.370-0.867)),patients with high CXCL8 gene expression have a better prognosis,and bioinformatics analysis of chemokine CXC subfamily genes is enriched for cell-cell signaling and receptor binding and metabolic pathways such as functional and chemokine signaling pathways.The possible mechanism of action of CXCL8 gene in COAD was analyzed by CXCL8 Gene set enrichment analysis and co-expression gene.Conclusion:The CXCL8 gene in the chemokine CXC subfamily has a high diagnostic value and is associated with the prognosis of COAD.This result needs to be further verified in the future.PART3:THE DIAGNOSIS AND PROGNOSTIC VALUE OF CXCL8,CXCL3 AND CXCL1 EXPRESSION IN COLONIC ADENOCARCINOMA OF THE FIRST AFFILIATED HOSPITAL OF GUANGXI MEDICAL UNIVERSITYObjective:To investigate the clinicopathological features and prognosis of CXCL8,CXCL3 and CXCL1 expression in colon adenocarcinoma.Materials and methods:We collected 38 pairs cancer tissue and adjacent normal intestinal tissue of colon adenocarcinoma patients to perform real-time RT-PCR;We collected of 212 tumor tissues and 47 adjacent normal tissues wax blocks of 212 cases of colon adenocarcinoma patients to use immunohistochemical examination and recorded general information and clinical pathological information of patients to analyze the expression of CXCL8,CXCL3 and CXCL1 genes with general information and clinical pathological information of patients by chi-square test.Univariate and multivariate analysis were used to know CXCL8,CXCL3,and CXCL1 gene expression correlated with patient prognosis.Results:Real-time RT-PCR and immunohistochemical results showed that the expression of CXCL8 has a high diagnostic value for COAD.The results of Multivariate analysis showed that CXCL8,CXCL3,CXCL1 protein expression and COAD patients' overall survival time were not statistically significant in COAD patients after adjusting TNM staging,tumor differentiation,tumor thrombosis and radical resection.Stratified analysis showed that CXCL8-positive patients had a longer overall survival in patients without adjuvant chemotherapy,and CXCL3-positive patients had shorter overall survival in patients with tumors less than 5 cm in diameter and with tumor thrombosis.Patients with CEA-positive patients had longer overall survival in CXCL1-positive patients.Finally,we included the clinical factors of CO AD patients in the nomogram model.The results showed that tumor TNM staging,tumor differentiation and whether the tumor had radical resection had the greatest impact on the prognosis of patients.Among the three genes CXCL8,CXCL3 and CXCL1,CXCL8 was the patient.The prognosis contributes the most.Conclusions:Our results suggest that CXCL8 may be a novel biomarker for the diagnosis and prognosis of COAD,and more samples and cohorts are needed for exploration and validation in the future.PART 4:ANALYSIS OF THE BIOLOGICAL FUNCTION OF CXCL8 IN SW620 AND RKO COLON CANCER CELL LINES USING SIRNA INTERFERENCE TECHNOLOGYObjective:To investigate the effects of siRNA silencing CXCL8 gene on gene expression,proliferation,migration,invasion,cell cycle and apoptosis of colon cancer cell lines SW620 and RKO.Materials and methods:The better siRNA targets for CXCL8 gene silencing were screened by transfecting two siRNA targets against CXCL8 gene into human colon cancer cell lines SW620 and RKO.The target CXCL8 gene of colon cancer cell lines SW620 and RKO were silenced,MTT assay was used to detect the proliferation of colon adenocarcinoma cell lines SW620 and RKO cells,and the cell migration ability was detected by scratch test.The number of cells passing through the transwell chamber was observed to detect the invasion and migration ability of the cells,as well as the cell cycle and apoptosis.Results:After siRNA-4 silenced the expression of CXCL8 gene in SW620 and RKO cell lines,MTT assay showed that the proliferation of colon cancer cell lines SW620 and RKO showed no significant change in SW620 proliferation ability,and RKO proliferative ability was significantly weakened;The results of scratch test showed that SW620 migration was significantly attenuated;Transwell method showed no significant change in RKO invasion,and migration was significantly weakened;experimental results on cell cycle showed no significant changes in SW620 cell cycle,but significantly blocked RKO cell cycle;The results showed a significant increase in apoptosis of SW620 and RKOConclusion:After silencing CXCL8 gene by siRNA,the apoptosis of SW620 cell line is increased,the proliferative ability of RKO cell line is weakened,the migration ability is weakened,the mitosis is slowed down,and the apoptosis is increased.