The Function Of EI24 In Inhibiting The Proliferation And Invasion Of Colorectal Cancer By Regulating CD44-mediated Oxidative Stress

Background:The proliferation and invasion of colorectal cancer are the main causes of patient death.Therefore,it is imperative to investigate the molecular mechanisms that drive its proliferation and invasion.A key player in this process is EI24,which is primarily found on the endoplasmic reticulum membrane.EI24 is essential for the formation of mitochondrial-related membranes,the promotion of autophagy formation,and the maintenance of normal cell metabolic stability.Tumor cells produce ROS mainly due to the high metabolism of mitochondria,endoplasmic reticulum,and cell membrane.The excessive accumulation of ROS can lead to oxidative damage in tumor cells.Moreover,it can stimulate autophagy of living mitochondria in tumor cells,which degrades intracellular catalase and further increases the accumulation of ROS.Ultimately,this process promotes apoptosis of tumor cells and inhibits tumor growth.ROS has the ability to reversibly oxidize crucial protein molecules in different signaling pathways,thereby regulating protein activity in a manner that is similar to post-translational modification.The PI3K/AKT signaling pathway is significant in regulating malignant phenotypes,including tumor proliferation,invasion,metastasis,and apoptosis.Alterations in tumor cell ROS levels can modulate the activity of pivotal protein kinases within the PI3K/AKT signaling pathway,thereby exerting regulatory effects on this signaling pathway.Several studies have demonstrated that tumor cells,particularly those with heightened metabolic activity in colorectal cancer,necessitate the establishment of protective antioxidant systems in a specific manner to eliminate excessive ROS in tumor cells and sustain cellular proliferation.Cluster of differentiation 44(CD44)is a cell adhesion molecule(CAM)that plays a crucial role in promoting tumor growth and inducing chemotherapy resistance.It stabilizes the glutamate cystine transporter on the surface of tumor cells,which in turn promotes the synthesis of GSH and inhibits the level of ROS in tumor cells.The interaction between EI24,CD44,and ROS has the potential to affect tumor proliferation and invasion,but further research is needed to understand the specific regulatory mechanism.This study aims to investigate the role and potential mechanisms of EI24 in the progression of colorectal cancer.Aims:1.Determine the expression of EI24 in colorectal cancer and explore the correlation between EI24 and clinical pathological features of colorectal cancer;2.Exploring the impact of EI24 on the malignant biological behavior of rectal cancer;3.Study whether there is a regulatory relationship between EI24 and CD44expression;4.Analyze the downstream molecular events of EI24 that mediate CD44 function and subsequently inhibit the proliferation and invasion of colorectal cancer;5.Construct a prognostic model for colorectal cancer based on EI24 and tumor related molecules through bioinformatics analysis.Methods:1.The expression level of EI24 in colorectal cancer cells was analyzed using colorectal cancer cell lines Caco-2,RKO,HT29,SW480,SW620,HCT116,HCT8,HT29,and immortalized normal intestinal mucosal epithelial cell line NCM460.Clinical samples and colorectal cancer tissue chips were used to detect the expression level of EI24 in colorectal cancer tissue,The relationship between the expression of EI24 and the clinicopathological characteristics of colorectal cancer and the clinical prognosis of colorectal cancer patients was analyzed,and the relationship between the expression of EI24 and the prognosis of colorectal cancer patients was further analyzed using the online database to explore the expression of EI24 in colorectal cancer and the relationship between EI24 and the clinicopathological characteristics and prognosis of colorectal cancer;2.A colorectal cancer cell model with stable overexpression and knockdown of EI24 was constructed through Lentivirus transfection.After the transfection effect was verified,the effects of EI24 high and low expression levels on the migration and invasion of colorectal cancer cell migration were further explored through plate cloning experiment,CCK-8 cell proliferation experiment,Transwell migration and invasion experiment,and nude mouse subcutaneous tumorigenesis model,The effects of high and low expression levels of EI24 on ROS and lipid peroxidation levels were detected using flow cytometry and oxidative stress assay.The effects of EI24 expression levels on the expression levels of key proteins regulating autophagy in colon cancer cells were detected using Western blot experiments and mitochondrial autophagy fluorescence experiments;3.Using the constructed colorectal cancer cell model with stable overexpression and knockdown of EI24,we continued to explore the expression of CD44 in colorectal cancer and whether the expression of EI24 can affect the expression and function of CD44 through bioinformatics analysis,protein immune coprecipitation(Co IP),mass spectrometry analysis,fluorescence co localization experiment,q RT PCR experiment,Western blot experiment and tissue microarray immunohistochemistry experiment;4.Western blot experiment was used to explore the correlation between the high and low expression of EI24 and the expression of key proteins in CD44 and PI3K-AKT signaling pathway.N-acetylcysteine(NAC),a ROS scavenger,was used to treat the previously constructed EI24 overexpression colorectal cancer cell line.Western blot experiment was used to verify the expression changes of key proteins in PI3K-AKT signaling pathway after clearing ROS.CCK-8 Transwell invasion and migration experiment and nude mouse tumorigenesis experiment were conducted to explore the regulation of tumor malignant phenotype after NAC treatment of EI24 overexpression cells.EI24 knockdown cells were treated with glutathione inhibitor Buthionine imide(BSO).The changes of ROS level and oxidative stress level of cells were detected by flow cytometry and oxidative stress kit;5.Bioinformatics analysis was performed to integrate relevant data from the TCGA dataset,and the abnormal expression of EI24,CD44,PIK3CA(PI3K),AKT1,AKT2,AKT3,MTOR,PTEN,and DNMT3 A in colorectal cancer was used to construct a colorectal cancer prognostic scoring system to evaluate the prognosis of patients with different clinicopathological characteristics and to assist in judging the effectiveness of targeted therapy and immunotherapy.Results:1.The m RNA and protein expression levels of EI24 in colorectal cancer cells are generally lower than those in normal intestinal epithelial cells;The expression level of EI24 in colorectal cancer tissue is significantly lower than that in adjacent tissues;Survival analysis shows that the expression level of EI24 is closely related to the prognosis of colorectal cancer patients,that is,the higher the expression level of EI24,the better the prognosis of patients;There is a significant correlation between the expression level of EI24 and tumor staging,meaning that patients with higher levels of EI24 expression have earlier tumor staging.2.The colorectal cancer cell line with EI24 overexpression and knockdown was successfully constructed by Lentivirus transfection;The results of plate clone formation experiment and CCK-8 cell proliferation experiment showed that overexpression of EI24 can inhibit the proliferation of colorectal cancer cells;Transwell migration and invasion experiments showed that overexpression of EI24 can inhibit the invasion and migration of colorectal cancer cells;The nude mouse tumor formation experiment suggests that EI24 can inhibit in vivo tumor formation in nude mice;Flow cytometry and lipid peroxidation detection showed that overexpression of EI24 can lead to the accumulation of ROS in colorectal cancer cells;The autophagy level test results showed that EI24 promotes autophagy.3.Using the constructed stable transfection EI24,CD44 molecules were screened through mass spectrometry analysis;Through bioinformatics analysis,it was found that the expression level of CD44 in colorectal cancer tissue was significantly higher than that in normal tissue;The results of q RT PCR,Western blot,and immunohistochemical staining showed a significant increase in the expression level of CD44 in colorectal cancer cells and tissues;Through Co-IP combined with mass spectrometry analysis,it was found that EI24 interacts with CD44;Fluorescence co localization experiments showed the co localization of EI24 and CD44;Bioinformatics analysis revealed a significant expression correlation between EI24 and CD44;QRT PCR and Western blot experiments found that EI24 can downregulate the expression of CD44.4.Transcriptome sequencing showed that PI3K/AKT signaling pathway was enriched after EI24 knockdown;Western blot experiments showed that EI24 regulates the PI3K/AKT signaling pathway by inhibiting CD44;Western blot experiments showed that after treating EI24 overexpressing cells with ROS scavenger NAC,the expression levels of photo PI3 K p85,photo AKT,photo m TOR,and DNMT3 A increased compared to EI24 overexpressing cells,while PTEN expression levels decreased compared to EI24 overexpressing cells.However,the expression levels of EI24 and CD44 did not show significant changes compared to EI24 overexpressing cells;The results of plate clone formation experiment,CCK-8 cell proliferation experiment,Transwell migration and invasion experiment showed that NAC treatment of EI24 overexpressing cells significantly increased the proliferation,invasion,and migration ability of cancer cells;The subcutaneous tumor formation experiment in nude mice showed a significant increase in the volume and weight of the tumor in the NAC treated group;The results of flow cytometry ROS detection and lipid peroxidation detection showed that after treating EI24 knockdown cells with GSH inhibitor BSO,the level of oxidative stress returned to elevated.5.Based on the experimental results in Part 4,9 colorectal cancer related genes(including EI24,CD44,PIK3CA(PI3K),AKT1,AKT2,AKT3,MTOR,PTEN,and DNMT3A)were screened and a quantitative colorectal cancer scoring system was constructed using bioinformatics analysis methods.The scoring system can effectively evaluate the prognosis of patients with different clinical and pathological characteristics,and has a significant correlation with target genes related to immunotherapy after targeted therapy,It has good clinical application value.Conclusion:In summary,our conclusion of this study is summarized as follows: In colorectal cancer tissue,the expression of EI24 is significantly reduced,and patients with higher levels of EI24 expression have earlier tumor staging and better prognosis;EI24 can affect the scavenging effect of CD44 on ROS by inhibiting the expression of CD44,promoting an increase in oxidative stress levels in colorectal cancer cells;On the other hand,changes in ROS levels can affect the expression of downstream PI3K/AKT signaling pathways,thereby inhibiting the proliferation,invasion,and migration of colorectal cancer cells;Finally,we constructed a tumor scoring system for colorectal cancer using EI24,CD44,and PI3K/AKT signaling pathway related genes.This scoring system can effectively evaluate the prognosis of patients with different subtypes of colorectal cancer,and exhibits significant correlation with targeted therapy and immunotherapy targets.