The MiR-103A-3P/TGFBR3 Axis Regulates TGF-β-Induced Orbital Fibroblast Activation And Fibrosis In Thyroid-Associated Ophthalmopathy

Background: Thyroid-associated ophthalmopathy(TAO)is a complex autoimmune disease whose pathogenesis has not been fully investigated,and orbital fibrosis is an important part of it.Orbital fibroblasts(OFs)are the most important effector cells in TAO.The development of innovative therapeutic options for the prevention of orbital fibrosis relies heavily on the study of the molecular mechanisms that regulate the activation of orbital fibroblasts during the course of TAO.micro RNAs(miRNAs)regulate the expression of target genes through enhanced mRNA degradation and inhibition of protein translation.Some miRNAs can activate OFs to influence the developmental process of TAO.However,the mechanisms involved have not been elucidated.Objective: To research the differential expression of miR-103a-3p in TAO patients,and to clarify that miR-103a-3p affects the expression of TGF-β/SMAD and ERK/JNK axes by targeting TGFBR3,thus promoting the fibrosis of GO-OFs and the progression of TAO disease.Methods: 1.Orbital adipose tissues from TAO patients and normal subjects were collected,primary orbital fibroblasts were extracted,cultured and identified,HE and Masson staining of the above orbital adipose tissues were performed to compare the histopathological differences between the two orbits;2.Differentially expressed miRNAs were obtained by bioinformatics methods;real-time fluorescence quantitative PCR(Quantitative Real-time PCR(QPCR)was performed to verify the expression of miRNAs;3.OFs were incubated with multiple concentrations of transforming growth factor β(TGF-β)under different time conditions,and the proliferation viability of OFs was detected by MTT assay,and the expression of miRNAs and fibrosis indicators Vimentin and Fibronectin within OFs was detected by QPCR to determine the GO-OFs cell model;4.Plasmids that overexpress or knockdown miRNAs was transfected into GO-OFs models,the expression levels of miRNAs and fibrosis indicators within GO-OFs was detected after transfection using QPCR,immunofluorescence(Immunofluorescence assay,IFA),and Western blot;5.Bioinformatic methods to screen miRNA target genes,QPCR,Western blot to verify the expression of target genes within lentiviral transfected cell models;6.Luciferase reporter assay(LUC)to verify the target binding relationship between miRNA and target genes;7.Plasmids and lentiviruses were transfected to construct overexpressed or null-loaded miRNAs and target genes into GO-OFs models.MTT,IFA,QPCR,Western blot were used to verify the expression of miRNAs,target genes and fibrosis indicators in the transfected cell models,and Western blot was used to detect the expression of downstream SMAD pathway and MAPK pathway-related factors.Results: 1.Significant fibrosis were visible in stained orbital adipose tissue sections of TAO patients.2.Eight miRNAs with high expression were screened after microarray analysis,and miR-103a-3p had the most significant expression upregulation.3.Cell proliferation viability,expression of Vimentin and Fibronectin of OFs increased after TGF-βincubation and correlated positively with the duration of TGF-β action.4.Plasmid transfection successfully overexpressed or knocked down the expression of miR-103a-3p within GO-OFs.In GO-OFs overexpressing miR-103a-3p,cell proliferation viability was increased and the expression of Vimentin and Fibronectin was increased.The opposite was true in GOOFs with knockdown of miR-103a-3p.5.From miRDB,Targetscan and miRPath DB prediction and screening by TAO Gene Chip GSE58331,combined with literature search,TGFBR3 was predicted to be the target gene of miR-103a-3p.TGFBR3 was significantly reduced in TAO orbital tissues.Lentiviral transfection of TGFBR3 overexpression significantly inhibited the proliferation viability of GO-OFs and downregulated the expression of Vimentin and Fibronectin,while sh-TGFBR3 knockdown promoted cell viability and the expression of Vimentin and Fibronectin.6.LUC assays showed that miR-103a-3p overexpression significantly inhibited WT-TGFBR3 3’UTR luciferase activity,and miR-103a-3p low expression promoted its luciferase activity.While no such effect was observed within the mutant phenotype.In tissues,miR-103a-3p expression showed a negative correlation with TGFBR3 expression.7.Overexpression of TGFBR3 significantly reduced the enhanced proliferation viability and increased expression of Vimentin and Fibronectin induced by miR-103a-3p overexpression in GO-OFs.miR-103a-3p overexpression significantly promoted the phosphorylation of ERK1/2,JNK,SMAD2 and SMAD3;while TGFBR3 overexpression partially inhibited these effects.Conclusion: MiR-103a-3p promotes the proliferative activity and expression of fibrosis-related proteins in OFs of TAO patients by targeting TGFBR3 to activate the downstream MAPK and SMAD pathways,and is a promoter in the pathogenesis of TAO,while TGFBR3 protein can slow down these effects.