EI24 Positive Feedback Regulates Autophagy To Inhibit Epithelial-Mesenchymal Transformation And Metastasis Of Colorectal Cancer

Objective:The high morbidity and mortality of Colorectal cancer(CRC)bring serious disease burden to the world.The etiology and pathogenesis of CRC are very complex,and its occurrence and development is a process of multi-factor,multi-step,internal and external interaction.Although a variety of CRC-related molecules have been found,the signal regulation network involved in the occurrence and development of CRC is complex,and the specific role and mechanism of these molecules have not been fully clarified.EI24(etoposide-induced gene 24)is a DNA damage response gene induced by etoposide in a p53-dependent manner.Its gene is located in 11q23-q24,a frequently mutated region of human cancer,and its mutation is closely related to malignant transformation and increased invasiveness of the tumor.EI24 can inhibit cell growth through p53-mediated apoptosis pathway and play an inhibitory role in many kinds of malignant tumors by inducing cancer cell apoptosis.At the same time,EI24 protein encoded by its gene is an important autophagy protein located in endoplasmic reticulum,which can promote autophagy flux by participating in the formation of autophagosome and play an inhibitory role in many kinds of malignant tumors.Studies have shown that the expression level and function of EI24 in different types of tumors are different.In majority of tumors,such as breast cancer and non-small cell lung cancer,the expression level of EI24 is lower than that in normal tissues,EI24 play its role as tumor suppressor gene by either inducing apoptosis,activating autophagy,inhibiting epithelial stromal transformation,or gene mutates itself.However,in skin cancer,the expression level of EI24 is higher than that in normal tissues,and it can promote the carcinogenesis of skin cancer and play the role of oncogenes.In pancreatic cancer,EI24 can play a dual role either inhibiting or promoting cancer.Although some studies have found that EI24 is related to the progress and metastasis of CRC,its specific role and mechanism has not been clarified.Methods:CRC cell lines with stable knock-down and over-expression of EI24 were established,and the construction efficiency was verified by qRT-PCR and Western Blot.The effects of EI24 on the following biological functions of CRC cells were detected:Cell counting kit-8(CCK8)was used to detect cell proliferation;Transwell test was used to detect cell migration and invasion;scratching test was used to detect cell migration;Flow cytometry was used to detect apoptosis.Then,the potential mechanism of EI24 in the invasion and migration of CRC was studied in the following aspects:The effect of EI24 on the formation of autophagosomes was observed by transmission electron microscope,the changes of apoptosis and EMT-related protein markers induced by EI24 were detected by Western Blot,and the expression changes of autophagy-related protein markers in CRC cells before and after starvation or autophagy inhibitor 3-MA were detected by Western Blot.Results:The cell model of stable knockdown of EI24(HCT116-shEI24 and HCT116shControl;LoVo-shEI24 and LoVo-shControl),the cell model of stable overexpression of EI24(DLD1-EI24 and DLD1-Control;SW480-EI24 and SW480-Control)were successfully constructed.CCK8 results showed that EI24 could significantly inhibit the proliferation of CRC cells(P<0.001),Transwell and scratch assay showed that EI24 could significantly inhibit the migration and invasion of CRC cells in vitro(P<0.001),and flow cytometry result showed that EI24 could significantly increase the apoptosis rate of CRC cells(P<0.001).The results of transmission electron microscope showed that the high expression of EI24 could significantly increase the number of autophagosomes in CRC cells,and the Western Blot results showed that the low expression of EI24 could increase the expression of anti-apoptosis protein Bcl-2 and decrease the expression of pro-apoptotic protein Bax and Bak.Moreover,the low expression of EI24 could reduce the expression of EMT-related factor E-Cadherin,while increase the expression of Snail,N-Cadherin and Vimentin,Conversely,the high expression of EI24 showed the opposite result.Moreover,in CRC,EMT mediated by EI24 and its inhibition on cell invasion and migration can be enhanced by starvingactivated autophagy,and this inhibition can be partially reversed by autophagy inhibitor 3-MA.Conclusions:In CRC,abnormally high expression of EI24 can reduce the proliferation,invasion and migration of CRC cells by promoting tumor cell apoptosis,autophagy and inhibiting the process of EMT.The potential mechanism may be that EI24 can regulate autophagy through positive feedback and inhibit the EMT process of CRC cells,which ultimately reduces the invasion and migration of CRC tumors.