| Colorectal cancer is the third most common cancer.Most colorectal cancers originate from precursor lesions such as adenomas and transform into adenocarcinoma.The whole process involves many biological behaviors,such as cell growth,cycle regulation,signal transduction,interaction between cells and extracellular matrix,angiogenesis and lymphangiogenesis,but the fundamental reason is the genomic instability of cells.Three known pathways leading to genomic instability in colorectal cancer are chromos-omal instability(CIN),microsatellite instability(MSI)and abnormal Cp G island methylation.CIN plays an important role in the occurrence of colorectal cancer,accounting for about 80%.Its molecular basis is the result of the interaction of oncogenes,oncogenes,cell cycle regulation genes and so on.Therefore,in order to improve the diagnosis and treatment level of colorectal cancer and improve the prognosis of patients,this study aims to integrate and analyze the gene expression data of colorectal cancer tumor tissue and adjacent normal tissue by bioinformatics and molecular biology techniques,and obtain the potential gene IMPDH1 related to colorectal cancer.The effects of IMPDH1 on the biological behavior of colorectal cancer were also explored,providing a research basis for the diagnosis and treatment of colorectal cancer.Part Ⅰ Screening potential genes related to the occurrence and development of colorectal cancer based on bioinformaticsObjective: Through bioinformatics,differentially expressed and differentially methylated genes between colorectal cancer tissues and normal tissues were screened,and their molecular mechanism in the occurrence and development of colorectal cancer was explored.Methods:1.Transcriptome and genome-wide DNA methylation Expression data sets were downloaded from Gene Expression Omnibus(GEO)database,and data analysis was conducted to identify differentially expressed genes and differentially methylated genes between colorectal cancer tissues and normal tissues.2.GO and KEGG function and protein-protein interaction(PPI)analysis were performed on differentially expressed genes and differentially methylated genes,and the mechanism of action of differentially expressed genes and differentially methylated genes was analyzed.3.The differential genes and differential methylated genes were correlated to clarify the influence of methylation on gene differential expression.Results:1.Through genomic data analysis,1958 differentially expressed genes were found between colorectal cancer tissues and normal tissues,including1025 up-regulated genes and 993 down-regulated genes,among which IMPDH1 was located in the top 50 of up-regulated genes.By analyzing DNA methylation data sets between colorectal cancer tissues and normal tissues,2661 differential methylation loci were obtained,including 2539 high DNA methylation loci and 122 low DNA methylation loci.Further analysis showed that differential DNA methylation loci correspond to 858 differential methylation genes,including 800 high methylation genes.58 hypomethylated genes.2.By GO functional analysis,it was found that differentially expressed genes and differentially methylated genes were significantly enriched in DNA replication and protein binding of cell function,sequence-specific DNA binding of biological process and adhesion of affinity cells by plasma membrane adhesion molecules,and cytoplasmic and plasma membrane components of cell components(P<0.01);KEGG functional analysis showed that differentially expressed genes and differentially methylated genes were significantly enriched in cell cycle,DNA replication,purine metabolism and neuroactive ligand-receptor interactions,calcium signaling,c AMP signaling,cell adhesion molecules,PI3K-Akt,Rap1 and other pathways,respectively(P<0.01).The differentially expressed gene IMPDH1 was significantly enriched in purine ribonucleotide monophosphate biosynthesis,cytoplasmic and cytoplasmic composition,active catalysis of cell function,and purine metabolic signaling pathway of KEGG function analysis(P<0.05).3.Through PPI network analysis,it was found that the genes with high Degree of interaction were SHMT2(Degree = 44,Up),FOXQ1(Degree = 19,Up)and TRIP13(Degree = 17,Up),MDFI(Degree = 16,Up);The proteins interacting with IMPDH1(Degree = 7,Up)include SKP2,PODXL,FTSJ2,CHEK1,ANKRD9,BSGNTL1,LRRC8 E,etc.4.Through association analysis of differential genes and differential methylated genes,it was found that some genes,such as down-regulated FBLIM1,GFRA2 and WNT2,were hypermethylated genes,up-regulated MYC and B3GNTL1 were hypomethated genes,and IMPDH1 was non-methylated genes.Conclusions: A large number of differentially expressed and differentially methylated genes between colorectal cancer and normal tissues were obtained from GEO database.These genes mainly affect the occurrence and development of colorectal cancer by influencing nucleo-plasma components,sequence-specific DNA binding,cell cycle,and neuroactive ligand-receptor interactions.Further analysis showed that IMPDH1 was a non-methylated gene and affected the development of colorectal cancer mainly through the biosynthesis of purine ribonucleotide monophosphate,the composition of nuclear plasma and cytoplasm,the active catalysis of cell function,and the purine metabolic signaling pathway.PPI protein network analysis showed that the proteins interacting with IMPDH1 included SKP2,PODXL,FTSJ2,CHEK1,ANKRD9,BSGNTL1,LRRC8 E,etc.Correlation analysis of differential genes and differential methylated genes suggested that DNA methylation may affect gene expression.Part Ⅱ Identification and clinical significance analysis of IMPDH1 and other related key genesObjective: The expression and clinical significance of key genes related to the development and progression of colorectal cancer were verified by The Cancer Genome Atlas(TCGA)dataset and real-time quantitative PCR.Methods:1.The TCGA dataset was used to verify the expression of differentially expressed genes between colorectal cancer tissues and normal tissues in colorectal cancer(including 478 colorectal cancer patients and 41 normal controls).2.Fluorescence quantitative PCR was used to detect the expression levels of up-regulated genes and down-regulated genes in colorectal cancer tissues.3.ROC curve and Kaplan-Meier(K-M)survival curve were drawn by R language to evaluate the diagnostic and prognostic value of these genes for colorectal cancer.Results:1.Up-regulated expression of FOXQ1,CDH3,CLDN1,SHMT2,TRIP13,MDFI and IMPDH1 genes related to the occurrence and development of colorectal cancer in TCGA data set(P<0.01).However,GFRA2,SCGN,BEST4,CXCL12 and CA7 were down-regulated(P<0.01),which was consistent with the result of gene sequencing.2.Fluorescence quantitative PCR showed that FOXQ1,CDH3、CLDN1and IMPDH1 were significantly up-regulated in colorectal cancer tissues(P<0.05),GFRA2,SCGN,BEST4 and CXCL12 were significantly down-regulated(P<0.05),which was consistent with sequencing results.3.ROC curve and K-M survival curve were plotted by R language,and the results showed that AUC of GFRA2,FOXQ1,CDH3,CLDN1,SCGN,BEST4,CXCL12,CA7,SHMT2,TRIP13,MDFI and IMPDH1 were all greater than 0.7.These results suggest that these genes have certain diagnostic value for colorectal cancer.However,BEST4,SHMT2 and TRIP13 were significantly negatively correlated with survival rate(P<0.05).Conclusion: Key genes in colorectal cancer were verified by TCGA database and q RT-PCR method,and it was found that FOXQ1,CDH3 and CLDN1、IMPDH1 were significantly up-regulated in colorectal cancer tissues,while GFRA2,SCGN,BEST4 and CXCL12 were significantly down-regulated,which was consistent with the results of bioinformatics analysis.ROC curve and K-M survival curve showed that GFRA2,FOXQ1,CDH3,CLDN1,SCGN,BEST4,CXCL12,CA7,SHMT2,TRIP13,MDFI and IMPDH1 might be potential biomarkers for colorectal cancer diagnosis.BEST4,SHMT2 and TRIP13 can be used as prognostic markers in patients with colorectal cancer.Part Ⅲ Effects of IMPDH1 knockdown on proliferation,invasion and migration of human colon cancer cellsObjective: To investigate the effects of IMPDH1 gene on proliferation,invasion and migration of human colon cancer cells.Methods:1.The expression of IMPDH1 gene in human normal colon mucosal cells FHC and human colon cancer cells HT29,SW620 and SW480 was verified by real-time quantitative PCR.2.The expression of IMPDH1 gene in human normal colon mucosal cells FHC and human colon cancer cells HT29,SW620 and SW480 was verified by real-time quantitative PCR.3.The effects of IMPDH1 gene knockdown on proliferation,invasion and migration of human colon cancer cells were analyzed by MTT,Transwell and Scratch healing experiments.Results:1.Real-time quantitative PCR analysis showed that IMPDH1 was expressed in both human normal colon mucosa cells and human colon cancer cells,and the expression level in human colon cancer cells was higher than that in human normal colon mucosa cells.2.After si RNA transfection of HUMAN colon cancer cells SW480 and HT29,the transcription and protein expression levels of IMPDH1 and related gene YB-1 were decreased(P<0.05).3.MTT assay showed that IMPDH1 knockdown inhibited the proliferation of HUMAN colon cancer cells SW480 and HT29 in vitro(P<0.05).4.Transwell results showed that IMPDH1 knockdown could inhibit the invasions of HUMAN colon cancer cells SW480 and HT29 in vitro(P<0.05).5.Scratch healing test showed that IMPDH1 knockdown inhibited the migration ability of SW480 and HT29 cells in vitro(P<0.05).Conclusion: IMPDH1 expression level in human colon cancer cells was higher than that in normal colon mucosa cells.Inhibition of IMPDH1 expression may inhibit the expression of YB-1 m RNA and protein.Inhibition of IMPDH1 inhibited the proliferation,invasion and migration of colon cancer cells in vitro. |
