A Competing Endogenous RNA Network Associated With The Pathogenesis Of Thyroid Associated Ophthalmopathy

Research purposeThyroid associated ophthalmopathy(TAO)is a potentially dangerous eye disease that threatens vision.TAO patients have clinical and pathological signs such as eyelid retraction,exophthalmos and soft tissue inflammation.TAO is an autoimmune cross reaction mainly involving thyroid and extraocular muscles.However,its pathogenesis is still unclear and further research and thinking are needed.Noncoding RNAs(nc RNAs),which are transcription products of DNA but do not encode proteins,can regulate both whole organisms and cells in various ways,such as via molecular functions(MFs)and by affecting biological processes(BPs).Nc RNAs include long noncoding RNAs(lnc RNAs),circular RNAs(circ RNAs)and micro RNAs(mi RNAs).Through numerous studies of disease-specific nc RNAs expression,its outstanding potential as new diagnostic and prognostic biomarkers has been identified.However,the biological role of nc RNAs in the formation and development of TAO is still unclear.Some nc RNAs show remarkable developmental stage specificity and tissue specificity in ocular tissues,suggesting that nc RNAs may play a role in the formation and development of TAO.In recent years,a novel hypothesis about lnc RNA/circ RNA-mi RNA-m RNA interactions has arisen,which is called the competing endogenous RNA(ce RNA)hypothesis.It is hypothesized that mutual interference occurs between both coding and noncoding RNAs through mi RNA response elements(MREs),forming an extensive regulatory network across the transcriptome.However,the ce RNA hypothesis is still a blank in the field of TAO.Moreover,some recent studies have shown that tRNA and its precursors are specifically cleaved into small fragments,called tRNA-related fragments(t RFs)by specific enzymes.t RFs interact with ribosomes and aminoacyl tRNA synthetase to regulate translation.However,these studies are still blanks in the field of TAO and need to be explored urgently.We will explore the role of nc RNAs in the pathogenesis of TAO from the following aspects:(1)TAO was treated with high-throughput sequencing technology.Non-coding RNAs(lnc RNA,circ RNA,mi RNA,t RFs)and m RNAs in orbital connective tissue of patients and normal persons were sequenced,and the differential genes were identified.(2)The protein-protein interaction network was constructed,hub gene was identified according to node degree,and mi RNAs related to differentially expressed lnc RNA/circ RNA and m RNA were predicted by seed sequence matching analysis.(3)Correlation coefficient analysis of screened lnc RNA/circ RNA was conducted to ce RNA network.(4)Bioinformatics analysis was performed on the obtained differentially expressed t RFs to predict their targeted genes and explore their roles in the pathogenesis of TAO.(5)Real-time quantitative reverse transcription PCR was performed for ce RNA network and differentially expressed t RFs highly correlated with the pathogenesis of TAO.(6)Draw the final conclusion.Research methods1.Three TAO patients undergoing orbital decompression were selected as the case group,and orbital fat /connective tissues of patients undergoing plastic surgery was used as the control group for high-throughput sequencing.2.Lnc/circ RNA,m RNA,and t RFs content of the samples were sequenced by chip and sequencer respectively.3.After normalization of the original data obtained by sequencing,differentially expressed genes were screened.4.Protein-protein interaction network was constructed and top 30 of hub gene with the highest node degree were screened.5.The mi RNAs associated with differential lnc RNA/circ RNA and differential m RNA were predicted by seed sequence matching analysis,and the targeted relationship pairs were found.The intersection of lnc RNA/ circ RNA-mi RNA relationship pair and m RNAmi RNA relationship pair was obtained.Correlation analysis was conducted for lnc RNA/circ RNA and m RNA in the intersection to find the effective lnc RNA/ circ RNAm RNA relationship pair.6.The downstream targeted m RNAs of the differentially expressed t RFs were predicted and intersected with the differentially expressed m RNAs obtained by sequencing to obtain the differentially expressed m RNAs that may be regulated by t RFs.7.Mi RNAs highly correlated with TAO pathogenesis were selected from the intersection mi RNAs obtained in step 5,and the effective lnc RNA/ circ RNA-m RNA relationship in their upstream and downstream was used to construct ce RNA network related to TAO pathogenesis.8.Gene Ontology enrichment analysis and KEGG Pathway enrichment analysis were performed for the differentially expressed m RNAs obtained in Step 6 that may be regulated by t RFs.9.Orbital connective tissue of 5 TAO patients after orbital decompression and 5orbital fat/ connective tissues of normal patients after plastic surgery were selected,and real-time quantitative PCR of reverse transcription was used.The existence of hub gene in ce RNA and its upstream lnc RNA /circ RNA,as well as the differentially expressed t RFs and its downstream targeted m RNAs that were closely related to the pathogenesis of TAO and previously sequenced were verified.Research result1.Through normalized processing,differential expression analysis and screening of differentially expressed genes,355 differentially expressed circ RNAs,242 lnc RNAs,361 m RNAs and 3 t RFs were screened out.2.By screening,five mi RNAs were picked out which were highly related with TAO pathogenesis and widely studied(mi R-21,mi R-27 a,mi R-27 b,mi R-146 a and mi R-155)from the predicted lnc RNA/circ RNA-micro RNAs relationship and m RNA-micro RNAs relations after the intersection of micro RNAs.3.The targeted lnc RNA /circ RNA and m RNA of the five mi RNAs mentioned above were used to construct the ce RNA network.4.GO and KEGG analysis were performed on the differentially expressed m RNAs in the ce RNA network.Based on the Gene Ontology functional enrichment analysis results,the differentially expressed m RNAs were mainly related to the BP categories biological regulation and cellular process,the CC category extracellular region,and the MF category signal transducer activity.Finally,pathway analysis of the differentially expressed m RNAs showed that multiple pathways were involved,suggesting that most of the differentially expressed m RNAs participate in the PI3K-Akt signaling pathway,extracellular matrix(ECM)-receptor interaction pathway and neuroactive ligand-receptor interaction pathway.5.GO and KEGG enrichment analysis were performed for the differentially expressed targeted m RNAs of t RFs,reflecting that the biological processes(BPs)of the target genes are mainly related to metabolic processes,biological regulation and cellular component organization.Regarding the cellular component(CC)category,cell parts and membrane parts were notable.For molecular function(MF),the target genes are related to protein binding,antioxidant activity,transcription factor interactions and translationregulated effects.According to KEGG pathway enrichment analysis,the majority of the potential target genes are involved in the m TOR signaling pathway,VEGF signaling pathway and glycosaminoglycan biosynthesis.6.The q RT-PCR results revealed that 3 m RNAs(CHRM3,CXCL1,FPR2)and 2lnc RNAs(LINC01820:13,ENST00000499452)were differentially expressed between the TAO group and the normal group,that is,two ce RNA regulatory pathways(LINC01820:13-hsa-mi R-27b-3P-FPR2 pathway and ENST00000499452-hsa-mi R-27a-3p-CXCL1)are likely to have potential regulatory roles in the pathogenesis of TAO.t RF5-GLUCTC and three m RNAs(PMAIP1,HSD17B2,ATF3)are differentially expressed in OFs of TAO patients.Research conclusionWe have constructed an innovative non-coding RNA ce RNA network in TAO,hoping to shrink the scope of potential therapeutic targets and candidate diagnostic biomarkers.In addition,we identified and verified two lnc/circ RNA-mi RNA-m RNA interaction pathways with high potential correlation with TAO pathogenesis in ce RNA network: namely,LINC01820: 13-hsa-mi R-27b-3p-FPR2 ce RNA pathway and ENST00000499452-hsami R-27a-3p-CXCL1 pathway,which may be involved in regulating inflammation and autoimmunity of TAO.Additionally,50 differentially expressed t RFs and 361 differentially expressed m RNAs were detected between the TAO group and the control group.Bioinformatic analyses showed that differentially expressed t RFs could regulate the m TOR/VEGF signaling pathway.In addition,based on q RT-PCR verification results,namely differential expression of t RFs and its target genes,we speculated that t RF5-GLUCTC may play a potential regulatory role in the adipogenesis reaction and fibrosis hyperplasia of OFs in TAO patients by targeting ATF3,HSD17B2 and PMAIP1.