| Colon cancer is a malignant tumor of digestive tract with high morbidity and mortality.Surgical resection is currently the first and most effective treatment for colon cancer.However,in advanced colon cancer,surgery cannot achieve radical resection,and postoperative metastasis and recurrence are easy to reduce the therapeutic effect and survival time of patients.Finding effective therapeutic targets for colon cancer is one of the key directions of colon cancer research.It is well known that metabolic reprogramming has become a major feature of cancer.One of the striking features of metabolic reprogramming is the abnormality of nitrogen metabolism.This study mainly focus on the changes of glutamine metabolism and urea cycle,two important nitrogen metabolism processes in colon cancer and their influence on the development of colon cancer.It is hoped that therapeutic targets with potential clinical significance related to glutamine metabolism and urea cycle can be found within the study.In the first chapter,36 genes related to glutamine metabolism and urea cycle were obtained.Three key genes related to prognosis of colon cancer,namely ASNS,CEBPA and CAD were screened out by Cox regression analysis using colon cancer data samples from TCGA database.It is also found that these three genes are all related to asparagine metabolism..Differential expression of these three genes in tumor tissue can be used to divide colon cancer patients into two clusters with distinct lifetimes.We further used immunohistochemistry,q RT-PCR and Western blot to detect the expression of 3 key genes in cancer tissues and matched paracancer colon tissues from 16 patients with colon cancer from China-japan Union Hospital of Jilin University to verify the cluster model.Moreover,bioinformatics analysis methods GO and GSVA were adopted to analyze the expression differences of biological function and metabolism-related pathways in the two clusters.The IPA gene interaction network system was then used to predict the upstream and downstream relationships and differential gene pathways of ASNS and CEBPA.Finally,XCell software and TIDE system algorithm were employed to analyze the difference of immune cell infiltration and the response effect to immunotherapy in the two clusters.The results showed that Cluster1 and Cluster2 were constructed through the expression differences of ASNS,CEBPA and CAD.The lifetime of Cluster1 is significantly higher than that of Cluster2.Through clinical specimens,it is preliminarily confirmed that the clustering model established is of practical clinical significance.Different expression patterns of ASNS and CEBPA genes were also found to be associated with TNM staging and lymph node metastasis of colon cancer,while the expression patterns of CAD were associated with metastasis and recurrence.The IPA molecular mechanism predicted that CEBPA is the upstream regulator of ASNS,and CREB,insulin and RNA Pol II are the key upstream factors regulating CEBPA and ASNS expression.In addition,TIDE algorithm analysis found that Cluster2 has a good response to immunotherapy.Overall,it is concluded that different expression patterns of ASNS,CEBPA and CAD can be used to predict the prognosis of colon cancer patients and the effect of immunotherapy.The main work in the first chapter is the predictive research based on bioinformatics methods,which has been verified by preliminary experiments.These results suggest that ASNS,CEBPA and CAD have the potential as novel new markers and therapeutic targets for colon cancer.But However,more detailed molecular biology experiments are further required.When we screened out tumor prognostic genes related to glutamine metabolism and urea cycle,we also screened out a gene CPS1 directly involved in the urea cycle in addition to identifying 3 key genes.Hence,we decided to focus on the role of CPS1 in colon cancer.Through experimental research methods of cell biology,molecular biology and metabonomics,this paper discusses the effects of CPS1 on the growth and proliferation of colon cancer cells,and further studies the possible molecular mechanism.Firstly,we used lentiviral transfection to knock out CPS1 gene in colon cancer cell line Hct116.Then,we studied the effects of knocking out CPS1 on the proliferation and migration of Hct116 cells through CCK8 experiment,cell scratch experiment,cell apoptosis,cycle experiment,and subcutaneous transplantation tumor experiment in nude mice.Finally,We studied the molecular mechanism and found out the upstream and downstream pathways of CPS1 by metabonomics and Western Blot experiment.The results showed that CPS1 knockout can enhance the proliferation,migration and tumor-forming effect of colon cancer cell line Hct116.Therefore,the preliminary conclusion can be drawn that CPS1 has an inhibitory effect on colon cancer cells.The differential expression of CPS1 changes the levels of some main intracellular metabolites such as nucleotides,amino acids and lipids.Furthermore,CPS1 plays an key role in inhibiting cancer by causing abnormal expression of AMP-AKT/AMPK-m TOR pathway and AMPK-FOXO1 pathway.In all,we elaborate the role of CPS1 in colon cancer from both macro and micro perspectives.CPS1,as a tumor suppressor of colon cancer,will certainly play an important role in the individualized treatment of colon cancer in the future.It also has the potential to become a molecular therapeutic target and prognostic marker of colon cancer. |
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