The Screening Of Pathological Gene And The Biological Function Research Of KIT Gene In Colorectal Cancer

Colorectal cancer is a commonly malignant tumor which has high morbidity and mortality,and the metastasis and recurrence is the main cause of patients'death.With the development of adjuvant chemotherapy and the multidisciplinary collaborative treatment,the diagnosis and treatment of colorectal cancer has significantly improved,but the prognosis of colorectal cancer patients with metastasis or recurrence is unsatisfactory.With the development of gene technology,exploring the key disease-associated gene in the process of development and metastasis of colorectal cancer by high-throughput technology can provide valuable biomarker of clinical diagnosis and treatment.Besides,investigating the biological functions of related genes can provide theoretical reference of tumor therapy.These issues are all hot research focus now.This doctoral thesis consists of the following there parts:1.The investigation of molecular diagnosis and molecular spectrum in colorectal cancerTissue and blood samples were collected from 36 patients with colorectal cancer in this study.After extracting DNA from these samples,we conducted target capture sequencing and bioinformatics analysis by Ion Proton high-throughput sequencing platform.We found that in 77.27%patients,the gene mutations were relatively consistent among tissue types.And the results of capture sequencing in blood samples can possibly provide reference to accurate diagnosis and medication guidance.In capture sequencing,we found that the genes with higher mutation frequency were TP53,KRAS,APC and KIT.Then,we assessed the correlation between the mutation status of KRAS and tumor staging in peripheral blood.Compared with KRAS wild type group,patients with KRAS mutation in peripheral blood have more mutation sites.Besides,KRAS mutation in peripheral blood has certain correlation with tumor stage and has higher clinical diagnostic value compared with the traditional clinical indicators CEA,CA199.2.The comparative study of difference in SNPs and CNVs between primary lesions and metastatic lesions in colorectal cancerA total of 6 samples were collected from 3 colorectal cancer patients with primary cancer lesions and lymphatic metastases.We analyzed the discrepant SNPs and CNVs by chip sequencing and bioinformatics analysis.Through the analysis of the GO and KEGG,we found that there is a certain difference in the biological function and signal transduction pathway of cell adhesion and angiogenesis between primary lesions and lymphatic metastases.Meanwhile,we found that KIT?gHR?IL3 and TSLP gene has coding change caused by discrepant SNP between primary colorectal cancer lesions and lymphatic metastases.Besides,we found and verified three discrepant CNVs between primary lesions and metastatic lesions(chr13:65798303-65883830;chr19:58540860-58570697;chr22:19966749-19985507).These three CNVs have significant differences between the primary and metastatic lesions of colorectal cancer,and these three discrepant CNVs may be biomarkers assessing the developmental stages of colorectal cancer.3.The biological function research of KIT gene in colorectal cancerIn previous studies,we found that the mutation of KIT gene has close relation with colorectal cancer,so we chose KIT gene to verify its relationship with colorectal cancer by cell function experiment.First,we compared the expression quantity of KIT gene between colorectal cancer tissue and para-carcinoma tissue by qPCR,and we found that the expression quantity of KIT gene increased significantly in colorectal cancer tissue compared to the para-carcinoma tissue.Then we knocked out the KIT gene by siRNA vector at cellular level and found that the proliferation ability of colorectal cancer cells was significantly decreased through CCK-8 and clone formation experiments.Besides,we also found that the invasion and migration ability of colorectal cancer cells reduced significantly by Transwell assay.And knocking out the KIT gene can promote the transform from mesenchymal to epithelial in colorectal cancer cells,resulting in a decline of cell migration ability.These results show that KIT gene is a potential target for the treatment of colorectal cancer.