The Mechanism Research Of Cysteine-rich Intestinal Proteinl(CRIP1)on Promoting Invasion And Metastasis Of Colorectal Cancer By Epithelial-Mesenchymal Transformation

Background and ObjectiveIn 2018,more than 1.8 million new cases of colorectal carcinoma(CRC)and 881,000 deaths occurred worldwide,accounting for about 10%of cancer cases and deaths.Overall,the incidence of colorectal cancer ranked third,but mortality ranked second.Due to changes in culture and eating habits,in most patients with colorectal cancer,it is asymptomatic at an early stage;the main cause of death in colorectal cancer patients is distant metastasis to other organs such as liver,lung,and brain.And bone,the prognosis is very poor.Over the past decade,our understanding of colon cancer characteristics has improved significantly.Despite significant advances in diagnosis and treatment,colorectal cancer(CRC)remains the main health burden worldwide,especially in developing countries with a Westernized lifestyle and an aging population.Therefore,the study of new molecular biomarkers(CRIP1)allows for the detection of colorectal cancer at an early stage or for the diagnosis and prognosis of colorectal cancer.Methods1.Fluorescence quantitative RT-PCR and Western blot were used to detect the level of CRIP1 in colorectal cancer cell lines.2.Immunohistochemical staining was used to detect the expression of CRIP1 in human colorectal cancer and normal intestinal mucosa.The expression of CRIP1 in metastatic colorectal cancer and non-metastatic colorectal cancer was compared.3.To construct the stable overexpression of CRIP1 SW620 and CRIP1 interfering CT116 colorectal cancer cell lines,and to verify the effect of CRIP1 on the proliferation of colorectal cancer cells by CCK-8 and in vivo tumorigenesis experiments in nude mice.4.Overexpression of CRIP1 in RKO cells interfered with CRIP1 expression in SW480 and HCT116 cells.The effects of CRIP1 on migration and invasion of colorectal cancer cells were verified by Transwell cell migration test and scratch test.5.Immunoblotting was used to detect SW480 cells interfering with CRIP1.It was proved that interfering with CRIP1 expression could inhibit the occurrence of EMT in colorectal cancer cells.6.The interaction protein AKT1 of CRIP1 was screened by immunoprecipitation and protein profiling,and the interaction between CRIP1 and AKT1 affected PI3K-AKT-mTOR signaling pathway to mediate the migration and invasion of colorectal cancer.Results1.Quantitative fluorescence RT-PCR and protein immunohistochemical staining showed that CRIP 1 expressed differently at the levels of RNA and protein in colorectal cancer cell lines.2.Immunohistochemical staining showed that CRIP1 was highly expressed in colorectal cancer,and the higher the expression,the later the stage.3.Overexpression of CREP1 can promote the proliferation of SW620 cells,interfering with CRIP1 can inhibit the proliferation of HCT116 cells.The tumorigenic rate and volume of mice injected with CRIP1 cells are significantly higher than those of control mice,while the tumorigenic rate and volume of mice injected with interference with CRIP1 cells are lower than those of control mice.4.Scratch test showed that the healing rate of over-expressed CRIP1 cells was significantly faster than that of fine-transfected control cells,and that of shNC cells was significantly faster than that of shCRIP1 cells.Transwell test showed that after over-expression of CRIP1,the invasive ability of colorectal cancer cells increased significantly,and after interference with CRIP1 expression,the invasive ability of colorectal cancer cells decreased significantly.Microscopically,SW480 cells interfering with CRIP1 were less invasive than shNC cells.5.CRIP1 could affect the expression of EMT-related proteins by Western blotting.6.The interaction protein AKT of CRIP1 was selected by immunoprecipitation and protein spectrum.It was verified that CRIP1 could affect the phosphorylation of AKT and activate PI3K-AKT-mTOR signaling pathway.Conclusions1.CRIP1 was expressed at both the level of mRNA and protein in colorectal cancer cell lines.2.Immunohistochemical staining showed that CRIP1 was highly expressed in colorectal cancer and its expression was high.It may be related to metastasis of colorectal cancer.3.Overexpression of CRIP1 can enhance the proliferation of colorectal cancer cells.4.Overexpression of CRIP1 enhanced the migration and invasion of colorectal cancer cells.5.Interference of CRIP1 expression can inhibit the occurrence of EMT in colorectal cancer cells.6.CRIP1 interacts with AKT to activate PI3K/Akt/mTOR signaling pathway and promote the proliferation,migration and invasion of colorectal cancer.