Screening And Verification Of Colon Cancer-Related T Cell Exhaustion Regulatory Genes Based On CRISPR Screening Technology

The incidence of Colorectal cancer(CRC)is at the top among all kinds of cancers,and the 5-year survival rate is significantly lower than the average level of cancer.Recently,many studies have shown that the immune microenvironment has a significant impact on the occurrence and development of cancer,and many immune checkpoint blockers have been developed.By changing the immune microenvironment,especially enhancing the activity of cytotoxic T cells,the treatment of cancer has achieved good clinical effects.However,for CRC patients,there is still a problem of low response population to immune checkpoint blockers,and even those who respond also have a high recurrence rate.Therefore,I hope to find key targets for intervention of CRC by analyzing the characteristics of colorectal cancer’s own cells and CRC immune microenvironment,and provide new ideas for future clinical treatment.In this study,experiments were carried out from three levels:the gene expression characteristics of CRC itself,the characteristics of infiltrating immune cells,and CRISPR screening animal model verification.Considering the heterogeneity of CRC,we focused our analysis on colon cancer in CRC.Firstly,the transcriptome data of colon cancer patients in TCGA were analyzed,and the results showed that there were a large number of differentially expressed genes between colon cancer samples and adjacent normal tissues.KEGG,GO and GSEA analysis further showed that cytokine expression and immunometabolism signaling pathways played an important role in colon cancer.These results suggest that colon cancer cells interact with infiltrating immune cells to jointly construct an inhibitory immune microenvironment.In order to further study the interaction between immune cells and colon cancer cells,the immune cell components in each colon cancer sample were predicted by bioinformatics,and the COX risk model was used to predict the prognosis of colon cancer.The results showed that CD8~+T cells were significantly correlated with the prognosis of colon cancer.Therefore,CD8~+T cells were mainly selected as the screening direction of immunotherapy.Considering that CD8~+T cells commonly exist in a dysfunctional depleted form in cancer,if genes expressed by colon cancer cells can significantly affect the depletion behavior of tumor-infiltrating CD8~+T cells,it will surely be effective to interfere with tumor growth.Therefore,we performed correlation analysis between the expression data of colon cancer samples and the proportion of infiltrating immune cells,combined with the prognostic data,to screen200 genes that may regulate T cell exhaustion and significantly affect the survival prognosis of patients.Further analysis revealed that these genes were mainly related to functions such as membrane-associated protein receptor binding,involvement in cellular REDOX and regulation of GTPase cycling.Considering the limitations of data analysis alone,a sg RNA library was constructed based on the selected genes.The potential target genes related to T cell exhaustion were further screened and verified by lentivirus-infected murine colon cancer cell lines and Crispr-Screen in immunocompetent/immunodeficient mice.In Crispr-screen screening,Trim33 and Mtus1 were significantly positively selected,and Dlx3 and Ruvbl1 were significantly negatively selected in immunocompetent mice.These selected genes were mainly related to microtubule organization center and nucleus.In immunodeficient mice,significant positive selection genes included Trim33 and Rp9,and significant negative selection genes included Snapc5,Ddx56,Zfp335,Eif2b5,Atp5d,Polrmt and Ruvbl1.These selected genes were mainly related to nuclear,RNA binding,protein binding and gene expression(transcription).Interestingly,Dlx3 gene(sg RNA)showed significant negative selection in immunocompetent mice(beta;p-value),but there was no significant selection tendency in immunodeficient mice(beta;p-value);Further data analysis showed that DLX3 gene had a significant effect on tumor stage,TP53mutation,pan-cancer expression and promoter methylation analysis results,and DLX3 high expression could significantly promote the depletion of CD8~+T cells(p-value).It may directly lead to an early onset of colon cancer and a poor prognosis(p-value).Therefore,this study suggests that DLX3 related signaling pathways and regulatory mechanisms may be of great value and significance,and provide theoretical basis for the research of immunotherapy targets for colon cancer in the future.