TRA2B Promotes Hepatocellular Carcinoma Progression By Inhibiting Ferroptosis Of HCC Cells And Regulating Immune Microenvironment Infiltration

Objective Hepatocellular carcinoma(HCC)is the third leading cause of cancer mortality worldwide,remains a major health concern.Further exploring the molecular mechanism of HCC,conducive to developing a novel therapy.Abnormal RNA binding proteins can lead to a range of adverse effects,eventually promoting the onset of human malignancies.Transformer 2 beta homolog(TRA2B)is an important RNA binding protein.This study aimed to investigate the role of TRA2 B in the development of HCC,provide new molecular target for predicting the prognosis of HCC,and provide rationale for the development of new treatment in clinical practice.Methods The RNA-seq data and corresponding clinical information of 371 HCC patients were obtained from The Cancer Genome Atlas(TCGA).R language was used to investigate the expression of TRA2 B difference between HCC and normal liver tissues.Data visualization were performed using ggplot2 package.At the same time,the correlation between TRA2 B and clinical characteristics of HCC patients was analyzed and the survival curve was plotted.HCC patients were collected after surgery in the Fifth Medical Center of PLA General Hospital.Differentially expressed genes were validated at m RNA and protein levels using q PCR,western blot and immunohistochemistry.Then,small interfering RNA and plasmid were used to knockdown or overexpression TRA2 B in HCC cells,and explored the effect of TRA2 B on the proliferation and invasion in HCC cell lines through CCK-8,Transwell and other experiments.In order to deeply explore the mechanism of the role of TRA2 B,the Linked Omics online database was used to analyze the co-expression genes of TRA2 B,and the volcano map was used to distinguish the positive and negative co-expression genes of TRA2 B.At the same time,GO and KEGG enrichment analysis on the positive and negative related genes of TRA2 B were performed using R package.The differentially expressed genes(DEGs)were obtained using R language and gene set enrichment analysis(GSEA)was performed for the DEGs.Analyze the correlation between TRA2 B and ferroptosis related pathways.Verify the occurrence of ferroptosis by detecting the levels of malondialdehyde(MDA),free iron,intracellular reactive oxygen species and glutathione in TRA2 B knockdown HCC cell lines.Finally,the infiltration of immune cells in HCC was analyzed by using R language and TIMER data.Results The results of qPCR and western blot showed that the expression of TRA2 B m RNA and the level of protein in HCC were significantly higher than that in the adjacent tissues.Shorter overall survival in HCC accompanies high expression of TRA2 B,suggesting that the abnormal expression of TRA2 B might be related to poor prognosis of HCC patients;The knockdown of TRA2 B in HCC lines decreased the ability of proliferation and migration.After GO and KEGG functional analysis of the positive and negative correlation co-expression genes of TRA2 B,it was found that the negative correlation genes of TRA2 B were involved in the regulation of the redox process of NAPDH products in the process of ferroptosis metabolism;At the same time,the results of GSEA enrichment analysis of DEGs showed that TRA2 B was negatively correlated with ferroptosis pathway,active oxygen product metabolism and glutathione metabolism,suggesting that there was a negative correlation between TRA2 B and ferroptosis in HCC,and the MDA,active oxygen and free iron in HCC cell lines with low TRA2 B were increased,while the GSH/GSSG ratio was significantly decreased,which further confirmed that TRA2 B could inhibit the occurrence of ferroptosis in HCC.The relationship between the expression of TRA2 B and the immune infiltration of HCC,we found that the abnormal expression of TRA2 B in Th2 cells(r=0.502,P<0.001),Thelper cells(r=0.432,P<0.001)and TFH cells(r=0.268,P<0.001)were positively correlated,and negatively correlated with DC cells(r=-0.248,P<0.001)and cytotoxic cells(r=-0.197,P<0.001),suggesting that the abnormal expression of TRA2 B affected the changes of the immune microenvironment of HCC.Conclusion In this study,we found that the abnormal expression of TRA2 B affect the prognosis of patients,and promote the progress of HCC by regulating ferroptosis and immune microenvironment of HCC,providing a novel therapeutic target for the treatment of HCC.