O-GlcNacylation Enhances Sensitivity To RSL3- Induced Ferroptosis Via The YAP/TFRC Pathway In Liver Cancer

Background:Ferroptosis characterized by iron-dependent accumulation of lipid hydroperoxides to lethal levels and a form of regulated cell death.YAP has been reported to play a key role in regulating ferroptotic death,and the YAP expression is increased and settled by O-GlcNAcylation.Nevertheless,whether O-GlcNAcylation can enhance the sensitivity of hepatocellular carcinoma(HCC)cells to ferroptosis is still unknown.Objective:The purpose of this research was to investigate whether O-GlcNAcylation increases the sensitivity of HCC cells to ferroptosis.Material and Methods:A protein expression change was detected by western blotting.Cell viability was detected by CCK-8.Lipid ROS were detected with C11-BODIPY dye.YAP protein localization was detected by Immunofluorescence.A protein interaction was detected by Co-immunoprecipitation.Chromatin immunoprecipitation was used to detect genes that interact with YAP.Target genes of YAP were detected by Dual Luciferase Reporter assay.Animal models were applied to detect effects of YAP on liver cancer.RNA-seq analysis was applied to analyse the expression changes of related genes.Results:In this study,we observed that PuGNAc hardly affected the production of lipid ROS and the level of MDA,the main end product of lipid peroxidation.PuGNAc can significantly enhance ferroptosis induced by RSL3.Over-expression of OGT results in global O-GlcNAcylation.Meanwhile,OGT overexpression enhanced RSL3-induced cell death,lipid ROS production and MDA production.In addition,PUGNAc contributes to iron mortality sensitivity,and its effects can only be reversed by ferrostatin-1.In addition,OGT knockdown inhibits global O-GlcNAcylation and RSL3-induced ferroptosis.Down-regulation of YAP eliminated ferroptosis and lipid ROS and MDA production aggravated by PUGNAc treatment.Further experiments confirmed that glycosylation did exist in YAP,and the o-GlcNAc site was mainly YAP Thr241.At the same time,knockdown of YAP had no effect on the viability of cells overexpressing YAP-T241 A with decreased or increased levels of lipid ROS and MDA.Subsequently,we identified 258 up-regulated genes and 276 down-regulated genes by reducing YAP expression and RNA sequencing(RNA-SEQ)analysis.When YAP was knocked down,TFRC expression was significantly reduced.In addition,bioinformatics analysis found that TFRC is an effective target of YAP.In addition,PuGNAc-induced global O-GlcNAcylation promotes YAP expression,resulting in increased TFRC expression at both protein and mRNA levels.However,this effect disappeared after YAP was suppressed.The results showed that YAP knockdown significantly inhibited iron accumulation and saved iron accumulation through TFRC overexpression.Meanwhile,TFRC overexpression significantly reduced YAP enhanced iron mortality sensitivity.In addition,by knocking down YAP and simultaneously overexpressing SLC7A11,we found that SLC7A11 did not save YAP-induced ferroptosis.Together,these results suggest that YAP enhances iron mortality sensitivity by regulating TFRC expression.According to the chip-Seq dataset,we identified TFRC as a valid target for YAP.When the-500 to-250 NT regions were deleted,the basal promoter activity of TFRC was reduced to a level similar to that of the pGL3 control vector.Meanwhile,the TFRC promoter activity was completely lost after transfection with del-YAP plasmid.Knockdown of YAP inhibited the activity of TFRC promoter and the overexpression of YAP-WT but not YAP-T241 A significantly promoted the activity of TFRC promoter.In addition,ChIP results demonstrated the existence of physical binding between YAP and TFRC promoters.In addition,PUGNAC-induced OGlcNAcylation enhances TFRC promoter activity.Inhibition of YAP eliminated PUGNAc enhanced TFRC transcription.Inhibition of TFRC also reversed OGT overexpression-mediated reduction in iron levels and susceptibility to ferroptosis.In addition,treatment with piperazine Erastin,a form of Erastin that increases in vivo stability,reduced the volume of xenografts derived from OGT overexpressed cells compared with xenografts derived from GFP-SH cells.Together,these results suggest that O-GlcNAcylation enhances ferroptosis sensitivity by modulating TFRC.Conclusion:In summary,we are the first to find that O-GlcNAcylation can increase ferroptosis sensitivity in HCC cells via YAP/TFRC.Our work will provide a new basis for clinical therapeutic strategies for HCC patients.