Identification And Validation Of Ferroptosis-Related Genes And Exploration Of Their Function In Thyroid Associated Ophthalmopathy

ObjectivesThyroid associated ophthalmopathy is an orbital autoimmune inflammatory disease that is commonly associated with thyroid dysfunction.Although the etiology of TAO is unclear,ROS accumulation and oxidative stress have been closely linked to the pathogenesis of TAO.Ferroptosis is an iron-dependent programmed cell death characterized by intracellular labile iron levels,excessive accumulation of reactive oxygen species and lipid peroxidation.Currently,there are few reports regarding the role of ferroptosis in TAO.This article aimed to identify ferroptosis-related genes with diagnostic and therapeutic potential in TAO and explore their relationship with immune cells and lnc RNAs.Materials and MethodsWe searched,analyzed,and screened the high-throughput gene datasets that related to TAO in the Gene Expression Omnibus database.GSE58331 was downloaded from GEO and selected for initial differential gene analysis and the identification of differentially expressed ferroptosis-related genes.Variance analysis of high-throughput genetic data was completed in R language.Samples that related to TAO from GSE58331,GSE185952 and GSE105149 were selected for receiver operating characteristic curve to the validation of the DE-FRGs.In vitro experiments,real-time quantitative polymerase chain reaction was used to verify the expression of DE-FRGs at the transcriptional level in OFs.CIBERSORTx algorithm was used to analyze the infiltration of 22 types of immune cells in TAO samples.In vitro,immunofluorescence experiments were conducted to further validate the expression of each characteristic molecules of macrophages in orbital tissues from TAO patients.Spearman and Person correlation analysis were employed to explore the correlation between DE-FRGs and immune cells and long Non-coding RNA respectively.TRRUST and DGIdb databases were used to construct gene-transcription factor regulatory networks and gene-targeted drug regulatory networks.Results1.Six DE-FRGs(ABCC1,CYBB,CTSB,TLR4,PEX2 and SLC38A1)were screened out in the dataset.All the six DE-FRGs were ferroptosis driver genes.ROC results showed that the AUC values of these genes were greater than 0.7 in at least two gene datasets,except TLR4,which imply a powerful diagnostic ability.The AUC value of PEX2 was greater than 0.7 in all gene datasets with its disease specificity and diagnostic power were strongest among six DE-FRGs.The outcome of RT-q PCR revealed that the expression of CTSB,PEX2,and ABCC1 was decreased in orbital fibroblast from patients with TAO2.The result of immune cell infiltrate analysis indicated increased infiltration of monocytes(P<0.001),macrophages M0(P=0.039),mast cells activated(P=0.008),and neutrophils(P=0.045)in orbital tissues from TAO patients.Meanwhile,mast cells resting(p=0.043)and macrophages M2(p=0.02)showed reduced infiltration in TAO samples.There were no gender differences in immune cell infiltration in the TAO patients.The results of immunofluorescence experiments showed a significant upregulation of the marker of M0 macrophage(CD68)and M1 macrophage(CD86)in orbital tissues from patients with TAO,and a significant decrease of the marker of M2 macrophage(CD206)which validate CIBERSORTx results for macrophage.Correlation analysis of FRGs and immune cells showed that ABCC1 had a significant strong positive correlation with resting mast cells and neutrophils(criteria for a significant strong correlation: the absolute value of Spearman’s R greater than 0.7 and correlation hypothesis test P value less than 0.05)and a significant strong negative correlation with plasma cells;CYBB had a significant positive correlation with M2 CYBB had a significant strong positive correlation with M2 macrophages and resting mast cells,and it had a significant strong negative correlation with monocytes and M2 macrophages;PEX2 had a significant strong positive correlation with resting mast cells.Two differentially expressed lnc RNAs,LINC01140 and ZFHX4-AS1,in TAO groups were identified as ferroptosis-related lnc RNAs.CYBB-LINC01140-TLR4,CYBB-LINC01140-SLC38A1,TLR4-LINC01140-SLC38A1,and CTSBZFHX4-AS1-CYBB may be potential RNA regulatory pathways in TAO.Targeted drugs and transcription factors for differential expressed FRGs were also screened out in our study.DiscussionSix DE-FRGs were identified and validated in this study,and they have powerful disease specificity.in the future,these gene could be used as molecular diagnostic markers for TAO or potential molecular targets for treatment.These six genes are iron death driver genes,and five of them are downregulated in orbital tissues of TAO patients.The trend in cellular experiments was consistent with the results of algorithmic analysis,and although there is no logically rigorous bioinformatics means to specifically manage the status of ferroptosis in each sample,this result may suggest a decrease of ferroptosis or the presence of iron death tolerance in orbital tissues from patients with TAO.This finding was very important to act as the supporting evidence of the study of ferroptosis in TAO in the future.Further genetic functional studies have revealed multiple potential mechanisms of action of DE-FRGs in the disease.