Mechanism Of Non-coding RNA In Modulating Orbital Fibroblasts Activation In Thyroid Eye Disease

Objective: This study aims to investigate the mechanism underlying the activation of orbital fibroblasts(OFs)in thyroid eye disease(TED)from the perspective of non-coding RNA(ncRNA)regulatory networks,in order to provide new biological targets for the diagnosis and treatment of TED.Methods:(1)Differentially expressed genes in TED and normal control(NC)orbital tissues from the GEO datasets were subjected to Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment annotation analysis to investigate their main metabolic pathways.(2)Orbital fat tissue samples from TED patients and NC volunteers were collected during surgery,subjected to HE and Masson staining to evaluate fibrosis.EGFR content and distribution were detected using immunohistochemistry(IHC)staining.Primary OFs were isolated and cultured from orbital fat tissues.Human microarray analysis was performed to select the differentially expressed micro RNA(miRNA).(3)The messenger RNA(m RNA)and protein of intercellular cell adhesion molecule(ICAM)-1,ICAM-4,vascular cell adhesion molecule(VCAM),CD44,fibronectin,α-smooth muscle actin(α-SMA),epidermal growth factor receptor(EGFR),protein kinase B(Akt)and p-Akt in orbital fat tissues were extracted and detected by real-time quantitative polymerase chain reaction(RT-q PCR)and western blotting(WB).(4)Pearson correlation analysis was used to examine the relationship between LPAL2 and ICAM-1,ICAM-4,miR-1287-5p expression levels,respectively.(5)TED OFs were treated with transforming growth factor-β1(TGF-β1)to investigate the effects of gradient concentration and time on the expression of LPAL2 and adhesion-related molecules in cells.(6)Transfection of small interfering RNA(si RNA)was used to knock down LPAL2 in OFs and human skin fibroblasts(HSFs).Mi R-1287-5p mimics or inhibitors were transfected into OFs and HSFs to produce overexpression or knockdown.(7)5-ethoxy 2-deoxyuridine(Ed U)and colony formation assays were performed to analyze cell proliferation;immunofluorescent(IF)staining was conducted to detect vimentin expression;and enzyme linked immunosorbent assay(ELISA)was used to detect the concentration of type I collagen in the culture medium.(8)The wild type(wt)and mutant type(mut)of LPAL2 or EGFR to miR-1297-5p were designed and cloned into the vector to obtain dualluciferase reporter plasmids,then transfected into 293 T cells.The luciferase intensity was detected using the dual-luciferase reporter assay system.The direct binding between miR-1287-5p and LPAL2/EGFR was further validated by the RNA-binding protein immunoprecipitation assay(RIP)and the RNA pull-down assay.(9)LPAL2 and/or miR-1287-5p gene expression were suppressed.The proteins involved in the PI3K/Akt pathway,adhesion molecules and fibrosis markers were evaluated using WB.The impact of LPAL2 and miR-1287-5p interaction on cell functions including cell proliferation and fibrosis were also investigated.Results:(1)KEGG pathway enrichment analysis revealed that differentially expressed genes between TED and NC orbital tissues were enriched in the focal adhesion signaling pathway.HE and Masson staining indicating severe fibrotic changes in TED orbital tissue.RT-q PCR and WB results demonstrated significant upregulation of m RNA and protein expression levels of adhesion molecules(ICAM-1,ICAM-4,VCAM,and CD44)in TED orbital tissue(P<0.01).(2)RT-q PCR results of TED and NC orbital tissue samples revealed that LPAL2 screened from the GEO datasets had the highest expression level in TED(P<0.005)and was positively correlated with the expression levels of ICAM-1 and ICAM-4.(3)Vimentin was visualized in primary orbital fibroblasts(OFs)isolated from TED orbital tissue by immunofluorescence(IF)showed higher flurescence intensity as well as the expression level of LPAL2 was significantly higher in OFs derived from TED(P<0.01).Treatment of TED OFs and HSFs with TGF-β1 in gradient doses and duration led to a significant increase of adhesion molecules,fibronectin,and LPAL2 expression levels(P<0.01).(4)Microarray analysis identified 8 differentially expressed miRNAs between TED and NC orbital tissues.Among these,miR-1287-5p expression level in TED orbital tissues and OFs was significantly lower than that in NC orbital tissues(P<0.005)and NC OFs(P<0.01),exhibiting a negative correlation with the expression level of LPAL2.Overexpression of miR-1287-5p surpressed lncRNA LPAL2 expression in both cell types(P<0.05),whereas miR-1287-5p knockdown significantly increased its expression(P<0.01).(5)Dual luciferase reporter assay showed that co-transfection of wtLPAL2/wt-EGFR and miR-1287-5p mimics into 293 T cells significantly inhibited luciferase activity(P<0.005),while it was significantly enhanced when co-transfected with miR-1287-5p inhibitors(P<0.01).RIP and RNA pull-down experiments showed a significant increase in LPAL2 expression level in the Ago2 group(P<0.01)and Bio-miR-1287-5p-wt group(P<0.01).(6)The KEGG pathway enrichment analysis of differentially expressed genes and predicted downstream target genes of miR-1287-5p in the GSE105149 dataset revealed significant enrichment in the phosphatidylinositol 3 kinase(PI3K)/Akt signaling pathway.IHC results showed more EGFR-positive regions in TED orbital tissues than NC(P<0.01).(7)Under the induction of TGF-β1,only knockdown of the LPAL2 in TED OFs significantly reduced the protein expression levels of key molecules(P<0.01).It also significantly reduced the Ed U positive cell proportion(P<0.01),number of colony formation(P<0.01),and type I collagen concentration in the culture medium(P<0.01).Conversly,knocking down miR-1287-5p alone yielded the opposite effect.(8)Simutaneous knocking down both miR-1287-5p and LPAL2,combined with the use of LY294002 to antagonize the PI3K/Akt pathway,result in a significant reduction in OFs activation and fibrosis(P<0.01).Conclusion:LncRNA LPAL2 competitively binds to miR-1287-5p,inhibiting its interaction with the downstream target gene EGFR and blocking the suppressive regulatory effect of miR-1287-5p on EGFR,leading to an elevation in adhesion molecules levels,thereby promoting the activation of TED OFs and fibrosis via the EGFR/Akt signaling pathway.