Whole Exome Capture Sequencing Identifies Candidate Genes Related To Colon Cancer

Objective:To explore the key genes and pathways in the progress of colon cancer, and reveal the genetic mechanism of the tumorigenesis and development of colon cancer systematically, we analyzed the genes in the tumorigenesis and development of colon cancer, and screened the mutant genes and their mutant site by using the Whole Exon Sequencing Technology. We also undertook the high throughput sequencing and Sanger sequencing technology combined with the analysis of functional genomics, protein interaction and signaling pathway database in our further experiment.Methods:1. We collected the colon carcinoma tissues and the paired normal colon tissue excised from surgical operation, extracted the genomic DNA by using DNA purification kit,and determined the quality and concentration of the DNA by trace spectrophotometer. Independent sequencing was done for each captured library by the next generation sequencing platforms of Illumina at Harvard medical school, US.2.Primers were designed to the corresponding gene mutations by using the Primer5, primer design software, according to the gene sequence information in the NCBI gene library; PCR was undertook to amplify the DNA after optimized the amplification conditions. The amplified DNA products were identified by electrophoresis, and those qualified products were sent to the genetic sequencing company BGI for Sanger sequencing;the sequencing results were transferred to nucleotide sequence map by Mutation Survary and Chromas, the mutants were determined by the comparison of amino acid coding sequence and the corresponding high-throughput sequencing results.3.The most differentially expressed genes were analyzed by GO term analysis, KEGG Pathway analysis and protein interaction analysis,in the aim to find the key genes in the the tumorigenesis and development of colon cancer and the pathways which they belong to.Results:1.851meaningless and missense mutations were found after the screening and analyzing of the genes in colon carcinoma tissues and the paired normal colon tissue by High-throughput Whole Exon Sequencing along with bio-medical softwares.2.Amongthe215mutations,PIK2B、CACNA、CAMK2A、FSHB、ITPR1、ITPF2、 ITPR3、MAP2K2、PLCB3were located in GnRH signaling pathway;while14genes include APC2、AXIN2、TCF7、TCF7L2、TGFBR2、TP53were located in7major signaling pathways in colon cancer pathway.Conclusions:Revealing the colorectal cancer tumorigenesis and development mechanism preliminarily by analyzing function,pathway and protein interaction of the genes with David, KEGG and STRING Laid the theoretical foundation for the early diagnosis, individual treatment and prognosis of colon cancer.