The Function And Mechanism Of Ubiquitin-like Protein FAT10 In The Progression Of Colorectal Cancer By Regulating Capn4 Expression

Background and Objective:Colorectal cancer(CRC)is one of the most common malignant tumors of digestive tract in clinic.Its incidence rate and mortality rate are the top three in malignant tumors.Currently,the overall survival rate and quality of life of patients have improved through surgical resection,chemoradiotherapy,targeted therapy and immunotherapy.However,due to the early symptoms of patients are easy to ignore,most of them are already advanced at the initial visit,and the pathogenesis of colorectal cancer is complex and diverse.Once metastasis occurs,their 5-year survival rate will significantly decrease.Therefore,exploring new therapeutic targets and molecular regulatory mechanisms for colorectal cancer will contribute to early diagnosis and intervention in patients with colorectal cancer,and provide a theoretical basis for the development of targeted drugs in the future.FAT10(Human HLA-F adjuvant transcript 10)is currently the only ubiquitin like protein that does not rely on ubiquitin and degrades its substrate directly through proteasome,playing an important role in the process of protein post translational modification.It has been reported that FAT10 is highly expressed in various malignant tumors,such as liver cancer,lung cancer and breast cancer.Its mechanism is different from that of the substrate protein binding,resulting in its different functions.If it is covalently bound,its binding protein will be degraded by proteasome pathway,while non covalently bound,it will inhibit the ubiquitination of the substrate protein to stabilize its expression.Although it has been reported that FAT10 promotes the proliferation of colorectal cancer cells through the degradation of P53 and promotes the degradation of ZO-1 through the autophagy lysosome pathway leading to colon cancer metastasis,considering the diversity of substrate modification methods of FAT10,the specific molecular mechanism of FAT10 in colorectal cancer needs further research and exploration.Methods:1.The expression of FAT10 in pan-cancer was explored through an online database.Further,fluorescence quantitative PCR,Western blot,and immunohistochemistry were used to detect the expression level of FAT10 in colorectal cancer tissues and corresponding adjacent tissues.The clinicopathological characteristics of colorectal cancer patients and the relationship between survival information and FAT10 expression were analyzed.2.Screening colorectal cancer cell lines,constructing FAT10 overexpression and knockdown stable transfection cell lines,and exploring the effects of FAT10 on the proliferation,invasion and migration of colorectal cancer cells through CCK-8,colony formation,wound-healing assays,transwell assays,subcutaneous tumorigenesis in nude mice and constructing lung metastasis models.3.Immunoprecipitation combined with mass spectrometry was used to identify possible binding proteins to FAT10 and identify Capn4 as a downstream research molecule.Fluorescence quantitative PCR and Western blot were used to verify the effect of FAT10 on the m RNA and protein expression of Capn4.The expression of FAT10 and Capn4 in colorectal cancer tissue was detected and the relationship between them was analyzed.Finally,rescue experiment was conducted to investigate whether Capn4 is a key molecule for FAT10 to promote the proliferation,invasion,and metastasis of colorectal cancer cells.4.The proteasome inhibitor MG132 was used to observe whether Capn4 was degraded through the proteasome pathway;Molecular simulation software simulates whether FAT10 and Capn4 dock,and immunoprecipitation verifies whether the two molecules bind;Change the expression of FAT10 after actinomycin CHX blocking protein synthesis to observe whether it affects the degradation rate of Capn4;After blocking the proteasome degradation function with MG132,observe whether FAT10 regulates Capn4.Further,observe the effect of changing the expression of FAT10 on the ubiquitination level of Capn4 by immunoprecipitation.Results:1.The online database found that the expression of FAT10 in liver cancer,lung cancer,breast cancer,bladder cancer,colorectal cancer and gastric cancer was significantly higher than that in normal tissues;Fluorescence quantitative PCR,Western blot,and immunohistochemistry confirmed that the expression of FAT10 in colorectal cancer was significantly higher than that in adjacent tissues;Analysis of clinicopathological features and survival information revealed that high expression of FAT10 was positively correlated with tumor invasion depth(T3+T4),tumor size(>5cm),and lymph node positivity,and patients with high expression of FAT10 predicted a worse prognosis.2.HCT116 and SW480 cells with relatively high expression of FAT10 and relatively low expression of DLD-1 were selected;The lentivirus constructed FAT10 knockdown and overexpression cell lines.In vitro cell experiments,including CCK-8,colony formation,wound-healing assays,and transwell experiments,confirmed that interfering with FAT10 expression significantly inhibited the proliferation,invasion,and migration of colorectal cancer cells,while overexpression of FAT10 promoted the proliferation,invasion,and migration of colorectal cancer cells;Animal experiments on subcutaneous tumorigenesis and lung metastasis in nude mice in vitro showed that the tumor size and lung metastasis number of FAT10 knockdown cell lines in nude mice were significantly lower than those in the control group,while the tumor size and lung metastasis number of over expressing FAT10 cell lines in nude mice were significantly higher than those in the control group.3.Immunocoprecipitation combined with mass spectrometry analysis technology uses Capn4 as a research molecule based on previous research and related literature reports by our research group;Western blot and fluorescence quantitative PCR showed that the protein level of Capn4 in FAT10 knockdown and overexpression cell lines decreased,while the protein level of Capn4 in FAT10 overexpression cell lines remained unchanged regardless of FAT10 overexpression or knockdown;Western blot was used to detect the expression of FAT10 and Capn4 in colorectal cancer tissues,and correlation analysis showed a positive correlation between FAT10 and Capn4;In the rescue experiment,it was found that upregulation of Capn4 in knockdown cell lines rescued the decreased in cell proliferation,invasion,and migration ability induced by downregulation of FAT10,while interference with the expression of Capn4 in FAT10 overexpression cell lines inhibited the increase in cell proliferation,invasion,and migration ability caused by FAT10 overexpression.This proves that Capn4 is a key molecule for FAT10 to promote the proliferation,invasion,and migration of colorectal cancer cells.4.The proteasome pathway is an important protein degradation pathway.When MG132 blocks the proteasome,the expression of Capn4 protein gradually increases,confirming that Capn4 is degraded through the proteasome pathway;Molecular simulation software found a docking between FAT10 and Capn4,and immunoprecipitation confirmed that FAT10 and Capn4 bind to each other;After actinomycin CHX blocks protein synthesis,overexpression of FAT10 can slow down the degradation rate of Capn4;Finally,under the action of MG132 blocking the proteasome,the protein level regulation of Capn4 by FAT10 was removed.Immunoprecipitation showed that the ubiquitination level of Capn4 decreased after overexpression of FAT10 and the ubiquitination level of Capn4 increased after downregulation of FAT10.Conclusions:FAT10 stabilizes the expression of Capn4 by inhibiting its ubiquitination,ultimately promoting the proliferation,invasion,and migration of colorectal cancer cells.