Annexin A4 Plasma Translocation In Colorectal Cancer And Its Molecular Mechanism

Background and purpose:Colorectal cancer（CRC）is one of the most common malignant tumors of digestive system with high mortality.Up to now,the exact pathogenesis of colorectal cancer is still unclear.A large number of literatures have shown that the pathogenesis of colorectal cancer is a multi-stage process involving multiple genes.Annexins（ANX）family is a calcium-dependent phospholipid binding protein superfamily,which plays an important role in the invasion,migration and chemotherapeutic resistance of various tumors.Annexin A4（ANXA4,A4）is a member of the Annexin family.A large number of studies have confirmed that Annexin A4 is significantly increased in colorectal cancer,and is involved in the invasion and metastasis of colorectal cancer.Our previous studies found that Annexin A4 expression was up-regulated in CRC tissues during phase Ⅰ-Ⅳ,and there was a phenomenon of translocation from cell membrane to cytoplasm in CRC.It has been confirmed that the aggregation ability of Annexin A4 membrane is regulated by calcium ions in normal cells.Therefore,we propose a scientific hypothesis that Annexin A4 may be translocated from cell membrane to cytoplasm in colorectal cancer cells.This phenomenon may be affected by Ca²⁺concentration.This study can provide important theoretical and experimental evidence for the development of CRC and targeted drug therapy.Methods:1.It is confirmed that Annexin A 4 is highly expressed in colorectal cancer cells and its expression is related to the clinicopathological characteristics of patients with colorectal cance:The expression of Annexin A4 in normal colorectal cells and different colorectal cancer cell lines was detected by Western blotting.The relationship between clinicopathological characteristics and Annexin A4 expression level in CRC patients was analyzed.2.Expanding the sample further to verify the abnormal expression of Annexin A4 in colorectal cancer and the phenomenon of serosal translocation:The distribution of Annexin A4 in normal colon cells,colon cancer cells,colorectal cancer tissues and adjacent tissues was detected by Western blots,immunofluorescence and immunohistochemistry.3.To observe the plasma translocation of Annexin A4 in colorectal cancer cell lines by adjusting the concentration of Ca²⁺:HT-29 cells were treated with different concentrations of calcium ion carriers and at different times to observe the effect of different concentrations and time of calcium ion reagents on Annexin A4 expression.Colorectal cancer cells were treated with calcium ion inhibitors（experimental group）,DMSO（control group）,membrane extraction protein and plasma protein,and then the expression of Annexin A4 was detected.Results:1.The expression of Annexin A was significantly increased in CRC cell line HT-29.The expression level of Annexin A was correlated with lymph node and distant metastasis,suggesting that the high expression of Annexin A4 could promote the invasion and metastasis of colorectal cancer.2.Western blots,immunofluorescence and immunohistochemistry showed that Annexin A4 was low expressed in normal colon cells,mainly in the cell membrane,but high expressed in CRC cells and mainly in the cytoplasm.3.Annexin A4 expression was the highest in colorectal cancer cells treated with 2M concentration and 24-hour calcium ion carrier,and was the best.Annexin A4 expression in HT-29 cells treated with calcium ion inhibitor was higher than that in DMSO group.Conclusions:1.Annexin A4 is highly expressed in colorectal cancer,and its high expression can promote the invasion and metastasis of colorectal cancer.2.Annexin A4 is translocated from the cell membrane to the cytoplasm in colorectal cancer,and is highly expressed in the cytoplasm.3.Annexin A4 in colorectal cancer cells is more likely to produce cytoplasmic aggregation after inhibiting calcium ion activity.