The Role And Mechanism Of Carbonic Anhydrase 2 In Ferroptosis Of Colorectal Cancer Cells

Colorectal cancer can reduce the incidence and mortality through early screening,surgical resection,adjuvant chemotherapy and targeted therapy.But it is still the third most common cancer in the world and the fifth most fatal cancer.Therefore,in-depth exploration of the pathogenesis and therapeutic targets of colorectal cancer,and the development of colorectal cancer predictive biomarkers will provide significant benefits to patients with early and advanced colorectal cancer.Carbonic anhydrase affects the occurrence,development and prognosis of colorectal cancer due to its specific expression pattern and regulation of the internal and external environment of tumor cells.Objective:To explore the expression of carbonic anhydrase 2(CA2)in colorectal cancer tissue and blood and its potential as a biomarker.To explore the effect of CA2on the development of colorectal cancer.To further investigate the role of CA2 in the tumor microenvironment of colorectal cancer.It lays theoretical foundation for CA2 as a clinical target for colorectal cancer.Methods:Differential expression factors of colorectal cancer were screened by paired colorectal cancer tissue and adjacent normal intestinal mucosal tissue proteomics and transcriptomics.The differential markers of potential colorectal cancer were further screened by paired patients’preoperative and postoperative serum proteomics.The relationship between CA2 and clinicopathological data was further verified by immunohistochemistry in tumor tissue and normal tissue of colorectal cancer patients.The expression of CA2 in serum was detected by ELISA in colorectal cancer patients and healthy controls.The effect of CA2 on the malignant phenotype of colorectal cancer was verified at the cell and animal level.The specific mechanism of the effect of CA2on colorectal cancer cells and tumor microenvironment was further explored and verified through transcriptomics,metabolomics and interacting proteomics.Results:The experimental results showed that CA2 was significantly down-regulated in colorectal cancer tumor tissue and significantly up-regulated in postoperative serum through multi-omics screening of colorectal cancer tissue and serum.The results confirmed that the expression of CA2 was down-regulated in colorectal cancer tissues,and the serum of patients with colorectal cancer,which had a good role in distinguishing colorectal cancer from normal healthy people.In addition,both in vitro and in vivo experiments demonstrated that knockdown/overexpression of CA2 could promote/inhibit colorectal cancer cell proliferation and metastasis,and inhibit/induce tumor cell apoptosis,which is a potential tumor suppressor factor.Further,by comparing the transcriptome and metabolome of the overexpression CA2 group and the control group,it was found that CA2 significantly affected the signal pathways and metabolic pathways closely related to ferroptosis.The detection of intracellular glutathione,malondialdehyde,reactive oxygen species,lipid reactive oxygen species and electron microscopy indicated that CA2 induced ferroptosis in colorectal cancer cells.Transcriptome GSEA analysis and q-PCR revealed that the p53-SAT1-ALOX15axis was significantly upregulated after CA2 overexpression,and inhibition of ALOX15 would partially complement CA2-induced ferroptosis.Immunoprecipitation found that CA2 interacted with p53 and proteins that regulate p53 stability.Western blott showed that overexpression of CA2 slowed down the degradation rate of p53.Through the intracellular p H probe,it was found that the intracellular p H decreased after the overexpression of CA2,and the ERK pathway was activated to promote the efficacy of the ferroptosis inducer Erastin.Through the analysis of the sc RNA-seq and bulk RNA-seq of colorectal cancer tissues,it was found that the proportion of CD8~+T cell infiltration was significantly increased in colorectal cancer tissues with high CA2expression.Through spatial reconstruction of CSOmap,it was found that tumor cells with high CA2 expression interacted more closely with CD8~+T cells,and the interferonγreceptor IFNGR2 on the surface of tumor cells with high CA2 expression was significantly up-regulated.Further co-culture of tumor cells with CD8~+T cells found that the killing ability of CD8~+T cells was inhibited/promoted by CA2knockdown/overexpression in tumor cells.In addition,combined with ferroptosis inhibitors,it was found that CA2 promoted the killing ability of CD8+T cells by inducing ferroptosis.Conclusions:1.CA2 is less expressed in the serum or cancer tissues of colorectal cancer patients than in the serum or colorectal tissue of health people,and the low expression of CA2 is significantly correlated with the poor clinicopathological characteristics and unfavourable prognosis of colorectal cancer patients.2.Knockdown/overexpression of CA2 in colorectal cancer cells can significantly promote/inhibit the growth and migration of tumor cells,suggesting the tumor suppressor function of CA2.3.CA2 promotes ferroptosis in colorectal cancer cells by inhibiting p53 degradation and activating the p53-SAT1-ALOX15 axis.4.CA2 activates ERK pathway by regulating intracellular p H and then affects the efficacy of ferroptosis inducer Erastin.5.CA2 affects the killing ability of CD8~+T cells in the tumor microenvironment by inducing ferroptosis of tumor cells.