| Colon cancer is a major malignant tumor of the digestive tract and is one of the most malignant tumors in the world.Colon cancer has a high recurrence and mortality rate.Despite improvements in surgery,chemotherapy,radiation and immunotherapy for colon cancer,survival rates remain low.Therefore,it is necessary to identify prognostic models that reliably predict clinical outcomes.Hypoxia is typically observed in the Tumor immune microenvironment（TIME）and is caused by insufficient oxygen supply.It has previously been reported that many transcription factors in tumor cells are activated under hypoxia conditions,thereby inducing downstream signals to regulate tumor angiogenesis and cell proliferation.There is a growing body of research on TIME,in which immune cells play a crucial role in the development of tumors.Interestingly,there is growing evidence of an association between tumor hypoxic features and tumor immunosuppression and immune escape.Receptors affected by hypoxia play an important role in the recognition and cleavage of a range of NK cell targets.Therefore,under hypoxia conditions,the killing ability of NK cells is reduced.Hypoxia can promote immuno-suppressive cells or immunosuppressive cytokines,which in turn block immune effector cells.Colitis-associated colon cancer was induced in wild-type mice,and hypoxia and T cell immunity in colon cancer tissues were analyzed,and it was found that hypoxia enhanced immunosuppressive effects by inhibiting CD4+effector T cells and promoting regulatory T cell activity.Tumor hypoxia is an important obstacle to the effective treatment of tumor.Therefore,studying the relationship between tumor hypoxia and immunity is helpful to improve the effect of immunotherapy.At present,the methods to study tumor hypoxia are limited.However,the existence of a public database of genetic and clinical data makes it possible to determine the relationship between hypoxia and prognosis.In this study,we downloaded data related to colon cancer from GEO and TCGA databases.Subsequently,hypoxic-related features were identified to predict prognosis and timing in patients with colon cancer.In addition,ANKZF1 was selected as a hypoxic-related gene for in vitro cell experiments to detect its effects on the proliferation,migration and invasion of 45 colon cancer cells.In this study,a new hypoxic risk model for colon cancer was identified based on 14hypoxic-related genes.This can not only predict the survival of colon cancer patients,but also assess TIME.The analysis of hypoxic relationship between prognosis and immune cells will help to improve the level of cancer treatment and prognosis of colon cancer patients.In addition,in vitro cell experiments showed that the expression of knockdown risk scoring model gene ANKZF1can inhibit cell proliferation,migration and invasion in colon cancer cells.In short,this study provides insights into the clinical value of hypoxic-related genes in colon cancer.Part One The prognostic characteristics of hypoxic-related genes inco-lon cancer and the construction of risk assessment modelObjective:Data related to colon cancer were downloaded from GEO and TCGA databases.Subsequently,we identified hypoxic-related features for predicting prognosis and TIME in patients with colon cancer.Methods:1.Select the subjects.Gene expression data and complete clinical annotations were obtained from two databases,the Gene Expression Database（GEO）and the Cancer Genome Atlas（TCGA）.2.Based on the expression data of 60 TCGA-colon cancer data sets,univariate Cox regression analysis was performed on hypoxic-related genes to screen out the genes associated with colon cancer prognosis（P<0.05）.These genes were then screened using Lasso-cox regression analysis in the glmnet package to reduce the number of genes in the risk score model.Based on the TCGA-colon cancer and GSE17536 data sets,Kaplan-Meier analysis was used to compare overall survival（OS）according to the risk scores of high-risk and low-risk subgroups using the Survival and survminer packages.ss GSEA algorithm was used to quantitatively analyze the relative abundance of TIME cells in each colon cancer sample according to the amount of TIME cell infiltration in the colon cancer sample in TCGA.Gene-set enrichment analysis was performed on the TCGA-colon cancer dataset using GSEA software to explore the underlying mechanisms behind the hypoxia-associated risk prognosis model.The prognosis model of colorectal cancer was established.3.All statistics were performed using R software（version 3.6.3,https://www.R-project.org）.Wilcox.test was used to screen statistically differentially expressed genes and infiltrating immune cells.When plotting Kaplan-Meier curves,significant differences in OS between groups are tested using logarithmic rank.Univariate and multivariate Cox regression analyses were also performed.P<0.05 was considered statistically significant.Results:This study downloaded colon cancer data from GEO and TCGA databases and identified a new hypoxia risk model to predict prognosis in colon cancer patients.Risk score is significantly associated with poorer overall survival of colon cancer and can be used as an independent prognostic factor for colon cancer.The study also found that hypoxia risk models predicted the immune microenvironment of colon cancer,and that high hypoxia risk scores were associated with increased immunosuppression and tumor purity,but with decreased immune activation and stromal infiltration.These results reveal the important effect of hypoxia on the immune response of colon cancer,and provide a simple and effective evaluation method for colon cancer patients.Part Two Study on the potential relationship between hypoxia andclinical immunotherapy of colon cancerObjective:Exploring the potential relationship between hypoxia and clinical immunotherapy of colon cancer is helpful to reveal the mechanism of hypoxia in the immunotherapy of colon cancer,and provide a new idea and basis for individualized immunotherapy of colon cancer.Methods:1.Based on the TCGA-colon cancer and GSE17536 datasets,six immune checkpoints were randomly selected to analyze their expression in the high-risk and low-risk groups.2.In order to identify the mutation status of high-risk and low-risk groups,somatic mutation data of colon cancer patients were downloaded from the TCGA-Colon cancer dataset for somatic mutation analysis.3.Based on the TCGA-colon cancer dataset,the genes with the top 20mutation frequencies between high-risk and low-risk patients were analyzed.4.Electron expression analysis was used to compare the expression levels of CA-IX and PD-L1 in colon cancer tissue samples and para-cancerous tissue samples to explore their association with hypoxia risk and immunotherapy response.5.The expression of CA-IX and PD-L1 in colon cancer tissue samples and paracancer tissue samples was detected by immunofluorescence.6.All statistics were performed using R software（version 3.6.3,https://www.R-project.org）.Wilcox.test was used to screen statistically differentially expressed genes and infiltrating immune cells.When P<0.05 is considered as a statistically significant difference.Results:1.Patients with high risk scores tend to suppress TIME by upregating immune checkpoints.2.Immune checkpoints such as PD1,PD-L1,CD80 and PDL2 were significantly up-regulated in high-risk groups.3.The expression of CA-IX was significantly increased in colon cancer,and the expression of PD-L1 in colon cancer tissues also showed an upward trend but was not significant.CA-IX and PD-L1 are significantly elevated in colon cancer tissues（Figures S1C and D）（P<0.05）.4.Compared with adjacent normal tissues,the number and degree of CA-IX and PD-L1 protein staining positive cells in colon cancer tissues showed an increasing trend.Part Three Knockdown of hypoxic-related gene ANKZF1 inhibits theprogression of colon cancerObjective:Different experimental methods were used to analyze whether knockdown of ANKZF1,a hypoxic-related gene,can inhibit the progression of colon cancer in vitro.Methods:1.Real-time PCR was used to detect the expression level of ANKZF1 in knockdown cells and screen out effective interference targets.2.CCK-8 kit was used to detect the cell activity of HCT116 in each group at 0 h,24 h and 48h.3.The proliferation of cells in each group was observed by plate clone formation assay.4.Cell migration and invasion ability of each group were detected by cell wound scratch test.5.Transwell cell migration and invasion assay was used to detect the cell migration and invasion ability of each group.6.Western blot analysis was performed to detect the protein expression levels of MMP2,MMP9,N-cadherin,e-cadherin and HIF-αin each group.7.All the experimental data were analyzed by SPSS22.0 software,and the measurement data were expressed as±s.The T-test method was used for both sets of data,and the p method was adopted.0.05 was considered statistically significant.Results:1.The expression level of ANKZF1 in knockdown cells was detected by real time-PCR to screen out effective interference targets.2.CCK8 detection showed that active colon cancer cells were significantly increased under hypoxia conditions,and the activity of cancer cells was inhibited in Si-ANKZF1 and Si-ANKZF1+1%O2 groups after ANZF1 knockdown.3.Plate cloning experiment showed that the proliferation of cancer cells was significantly increased under hypoxia conditions,and the proliferation of cancer cells in Si-ANKZF1 and Si-ANKZF1+1%O2 groups was inhibited after ANKZF1 knockdown.4.The cell wound scratch test showed that the migration of cancer cells was significantly increased under hypoxia conditions,and after ANKZF1 was knocked out,the migration of cancer cells in Si-ANKZF1 and Si-ANKZF1+1%O2 groups was inhibited.5.The results of Transwell cell migration and invasion experiments showed that the invasion of cancer cells was significantly increased under hypoxia conditions,and the invasion of cancer cells in Si-ANKZF1 and Si-ANKZF1+1%O2 groups was inhibited after ANKZF1 knockdown.6.Western blot analysis was performed to detect the protein expression levels of MMP2,MMP9,N-cadherin,e-cadherin and HIF-αin each group.Conclusions:1.This study downloaded colon cancer data from GEO and TCGA databases and identified a new hypoxia risk model to predict prognosis in colon cancer patients.Risk score is significantly associated with poorer overall survival of colon cancer and can be used as an independent prognostic factor for colon cancer.The study also found that hypoxia risk models predicted the immune microenvironment of colon cancer,and that high hypoxia risk scores were associated with increased immunosuppression and tumor purity,but with decreased immune activation and stromal infiltration.These results reveal the important effect of hypoxia on the immune response of colon cancer,and provide a simple and effective evaluation method for colon cancer patients.2.Immune checkpoints PD1,PD-L1,CD80 and PDL2 were significantly up-regulated in the high-risk group.Therefore,identifying the underlying mechanisms and immune checkpoints of hypoxia in immune escape may help identify potential therapeutic targets for colon cancer.This study also demonstrated that CA-IX expression is up-regulated in colon cancer tissues,which further suggests that hypoxia plays an important role in mediating the progression of colon cancer.3.Knocking down the expression of ANKZF1 can inhibit the proliferation,migration and invasion of colon cancer cells,and it also indicates that ANKZF1 may play an important molecular regulatory role in the progression of colon cancer. |
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