Mechanism Of Interaction Between MIF And SLC3A2 To Regulate Ferroptosis And Promote Colorectal Cancer Development

Colorectal cancer(CRC)is one of the most dangerous gastrointestinal malignancies affecting human health.CRC is the third most common cause of cancer death in both men and women in the United States.And in our country,CRC morbidity and mortality ranked third and second,respectively,CRC tends to increase and appear younger with factors such as lifestyle and dietary changes in people.CRC occurs as a result of intestinal epithelial cells becoming cancerous due to a number of internal and external factors.Chronic inflammation is considered an important factor in CRC development,and elucidating the role of inflammatory factors in CRC development may lead to new ideas for CRC treatment.Our research group members has proved that MIF can promote the chemotaxis and growth of pre-metastatic CRC cells in the liver,colorectal cancer cells were coated in the upper chamber of transwell and HHSEC or HUVEC were cultured in the lower chamber,they had found that HHSEC-induced migration of colorectal cancer cells was 3.14 times higher than in the HUVEC group.MIF is a multipotent cytokine that plays an important role in inflammation,cell proliferation,angiogenesis and tumorigenesis.Multiple studies have shown that MIF plays an important role in the development of several types of malignant tumors,including breast,bladder,and gastrointestinal cancer.This research is based on the previous experiment of the our group,we had detected the expression of MIF proteins in CRC and analyzed the association with clinicopathological features,further screening and validating MIF interacting proteins by proteomic methods.The effects of MIF and the interacting protein SLC3A2 on CRC proliferation and migration are discussed,and the effect of MIF on ferroptosis in CRC cells via SLC3A2 was investigated.It provides some ideas for the development of personalized therapy and targeted drugs in CRC.METHEODS1.The expression of MIF in CRC fresh tissues as well as CRC cell lines were detected by qRT-PCR and Western blotting.The expression of MIF in paraffin tissues and matched normal tissues of CRC were detected by immunohistochemistry,and analyzes the correlation with clinical pathological features.2.The interaction protein SLC3A2 of MIF was screened by GST-pull down,immunoprecipitation,mass spectrometry and biological information methods.3.Biological effects of shMIF and shSLC3A2 on CRC cells were detected by CCK-8 assay,transwell migration assay,and immunofluorescence after transfection with shMIF and shSLC3A2 in SW480 and SW620 cells.4.Biological effects of shMIF and shSLC3A2 on ferroptosis in CRC cells were measured.GPX4 expression levels were detected by immunofluorescence,qRT-PCR and Western blotting;reactive oxygen species(ROS)status was detected by flow cytometry;ratio of reduced glutathione(GSH)to oxidized glutathione(GSSG)was detected by ELISA,and mitochondrial morphology of CRC cells was observed by transmission electron microscopy(TEM).5.In vivo,the tumor growth rate and volume was monitored by subcutaneous tumor assays in mice,and biological changes in cell proliferation,EMT phenotype,and iron death-related markers were measured by qRT-PCR,Western blotting,immunohistochemistry and ELISA.RESULTS1.Expression of MIF in CRC cells and cancerous tissues increases and is associated with clinical pathologic features.2.Silencing MIF expression inhibits the proliferation and migration of CRC cells.3.SLC3A2 is one of the interacting proteins of MIF.4.SLC3A2 expression was increased in CRC cells,silencing SLC3A2 alone or MIF+SLC3A2 inhibits proliferation,migration,and epithelial mesenchymal transitions(EMT)in CRC cells.5.Silencing MIF or/and SLC3A2 expression promoted ferroptosis in CRC cells.6.In vivo experiments showed that silenced MIF or SLC3A2 inhibited the tumour growth in mice,and promoted the ferroptosis of tumor cells.CONCLUSIONMIF interacts with SLC3A2 to promote the development of colorectal cancer through the ferroptosis mechanism.The innovation of this study:1.SLC3A2 was proposed as an interacting protein of MIF;2.By interacting with SLC3A2,MIF inhibits the ferroptosis in CRC.