IFN-? Inhibits The Growth Of Liver Cancer Cells Through TRIM21-mediated Oxidative Stress

TRIM21 is a member of the TRIM family,due to the inclusion of the RING domain,it has the activity of the E3 ubiquitin ligase and is capable of ubiquitylating a wide variety of substrates and therefore has a wide range of functions.Multiple proteins of the TRIM family are reported to be closely related to cancer,previous studies mainly focused on TRIM21 mediated autoimmune disease,the antiviral response,and the regulation of interferons production,etc.TRIM21 has been reported to have decreased expression in cancerous tissues such as hepatocellular carcinoma and diffuse large B lymphoma?DLBCL?,suggesting that TRIM21 may play an important role in the process of cancer.However,the specific mechanisms and signaling pathways associated with cancer are still unknown.Our previous animal experiments showed that TRIM21 can regulate intracellular ROS levels through the p62/Keap1/NRF2/ARE pathway,and TRIM21-deficient mice are more resistant to strong oxidants such as arsenic trioxide?As₂O₃?.Interferon has a dual role of anti-virus and anti-tumor,and is widely used in clinical,IFN-?has been shown to have inhibitory effects on a variety of tumor cells,but its mechanism of action on hepatocellular carcinoma has not yet been fully elucidated,further research on its mechanism will help better use of IFN-?for clinical treatment of liver cancer and other diseases.Separate studies have shown that IFN-?can promote TRIM21expression and IFN-?promote intracellular ROS production,which is a key step in the efficacy of IFN-?.At the same time,based on the role of TRIM21 in regulating intracellular ROS levels,there is an inseparable relationship between IFN-?,TRIM21and ROS.However,there is no direct evidence to indicate that TRIM21 is involved in the regulation of IFN-?-induced increase in intracellular ROS levels and inhibition of hepatoma cell proliferation,Further elucidation of the relationship between IFN-?and TRIM21 and the mechanism of action is beneficial to provide theoretical basis for the diagnosis and treatment of the hepatocellular carcinoma for IFN-?and TRIM21.Objective:Using the data in the TCGA database to compare the expression of TRIM21between cancer and adjacent tissues,analyze the effect of TRIM21 expression on the prognosis of patients with liver cancer,and evaluate the role of TRIM21 in hepatocellular carcinoma.To further clarify whether IFN-?can inhibit the proliferationofhepatocellularcarcinomacells,andwhetherthe TRIM21/p62/Keap1/NRF2/ARE pathway is involved in the regulation of oxidative stress of hepatoma cell lines by IFN-?,to lay a foundation for the evaluation of TRIM21 in clinical application,and to provide a theoretical basis for the application of IFN-?in the treatment of liver cancer.Methods:Firstly,TRIM21 gene expression data and clinical data were download from the TCGA database on the UCSC website?http://xena.ucsc.edu/?,then compare the expression of TRIM21 between cancer and adjacent tissues.Based on the low-to-high ranking of TRIM21 expression in cancer tissue samples,the samples were divided into three groups.The survival analysis of the three groups of samples was performed to compare the 5-year recurrence-free survival?RFS?and overall survival?OS?,simultaneous analysis of the association between the expression level of TRIM21 and clinical pathological data.Secondly,the SMMC-7721 cell line was used as a model to observe the inhibitory effect of IFN-?on hepatoma cells.The expression of TRIM21 protein and the production of ROS were detected after IFN-?treatment.After the cells were transfected with the shTRIM21 and shNC lentiviruses,the difference in cells treated with IFN-?was compared to determine whether TRIM21 was involved in the process of IFN-?inhibiting the growth of hepatoma cells.Immunoblotting,immunofluorescence,and RT-qPCR methods were used to detect the changes of anti-oxidation-related proteins after IFN-?treatment and the differences between shTRIM21 and shNC cells,to determine whether TRIM21 is involved in the inhibitory effect of IFN-?on hepatoma cells through the p62/Keap1/NRF2/ARE pathway.Results:1.The analysis of TCGA database data has not been able to clarify the differences in the expression of TRIM21 in hepatocellular carcinoma and adjacent tissues.But the 5-year recurrence rate of TRIM21 low-expression group was higher than that of medium and high expression group,however,there was no significant difference between medium and high expression groups.At the same time,there was no significant difference in overall survival between the low,medium and high TRIM21expression groups.2.The expression of TRIM21 is negatively correlated with the TNM stage of tumors,and patients with a history of hepatitis are more likely to have high expression of TRIM21.3.IFN-?can inhibit the proliferation of hepatoma cells and is dose-dependent within a certain concentration range.4.The inhibitory effect of IFN-?on TRIM21 knockdown hepatoma cells was reduced,indicating that TRIM21 is involved in the inhibitory effect of IFN-?on hepatoma cells.5.IFN-gamma can induce the expression of TRIM21 and promote the production of intracellular ROS.6.In the cells transfected with shTRIM21,there were more p62-dimer active forms after treatment with IFN-?,more degradation of Keap1,increased NRF2translocation into the nucleus,and increased expression of NRF2 regulated genes HMOX1 and NQO1.At the same time,combine with the experimental result that the inhibitory effect of IFN-?on the cells transfected with shTRIM21 was weakened,it was shown that the TRIM21/p62/Keap1/NRF2/ARE pathway is involved in the inhibitory effect of IFN-?on hepatoma cells.Conclusions:The expression of TRIM21 is negatively correlated with recurrence after radical resection in patients with liver cancer and TNM stage.TRIM21 may be a tumor suppressor gene in liver cancer cells and participates in the development of liver cancer.Therefore,TRIM21 may become a diagnostic or predictive marker for liver cancer,and may also become a target for clinical treatment.By comparing the shTRIM21 and shNC cells,it was found that IFN-?can inhibit the proliferation of hepatoma cells by increasing the intracellular ROS levels,and TRIM21 can negatively regulate anti-oxidation-related proteins through the p62/Keap1/NRF2/ARE signaling pathway,increase the sensitivity of liver cancer cells to IFN-?.Our discovery has the following important implications:First,it provides a theoretical basis for the anti-tumor effect of IFN-?,and provides a new choice for the treatment of liver cancer.Secondly,the anti-oxidation negative effect of TRIM21 can be used to combine with traditional radiotherapy and chemotherapy to improve the therapeutic effect of liver cancer.