The Role Of MiR-146a In Regulation Of Orbital Fibroblasts In Thyoid–associated Ophthalmopathy

Thyroid-associated ophthalmopathy(TAO),also known as Graves ophthalmopathy(GO),is a complex autoimmune disease that is closely related to Graves disease(GD)and is the most common GD.The most important damage to the extrathyroid organs.It is disfiguring and potentially blinding,eventually leading to remodeling of the eyelid tissue and destruction of adjacent eye structure.The exact pathogenesis is still unclear.There is currently no specific treatment for the treatment of TAO in the clinic.Insulin-like growth factor-1 receptors(IGF-1R)and thyroid stimulating hormone receptors(TSHR)are currently two of the two autoantigens associated with TAO.The orbital fibroblasts(OFs)express the insulin-like growth factor-1 receptor(IGF-1R),and the TSHR antibody immunoprecipitation complex can effectively act on IGF-1R to form a physical and functional signaling complex with IGF-1R.Some studies have shown that inhibition of IGF-1R activity leads to a decrease in signaling initiated at either receptor.These experimental findings serve as a recently completed monoclonal inhibitory antibody to teprotumumab that targets IGF-1R in TAO.The basic principles of the test provide the basis.The results of the trial in active,moderate to severe disease showed a dramatic decline indisease activity and severity,and the results were encouraging.Recent animal model experiments have further confirmed that IGF-1R plays a key role in the pathogenesis of TAO.MiRNA-146 a has multiple target genes and is a typical multifunctional miRNA.It plays its characteristic regulatory role in the same cell pathway according to different conditions,and participates in immune regulation,cell proliferation and differentiation,apoptosis,and extracellular matrix metabolism.Wait for a variety of life processes.Our previous study has found that miRNA-146 a is lowly expressed in sputum tissue and peripheral blood mononuclear cells(PBMC)of TAO patients,but highly expressed in normal human eyelid tissue.In addition,IGF-1R is the target of miR-146 a revealed by Bioinformatics analysis using TargetScan 6.2(http://www.targetscan.org,released June 2012).Under the background conditions of the above research,we conducted a basic research on the orbital fibroblasts of TAO.Objective:We will start from the new viewpoint of miRNA to explore the regulation of miR-146 a on the morphology and proliferation of orbital fibroblasts in thyroid-associated ophthalmopathy,and to elucidate the role of miRNA-146 a in IGF-IR and its downstream molecules in orbital fibroblasts.The differential expression of Akt protein and related inflammatory factor TNF-? plays a foundation role in the study of the pathogenesis of TAO in orbital fibroblasts.Exploring the pathogenesis of TAO at the genetic level,and researching targeted therapeutic drugs for TAO,laying the foundation for the prevention and treatment of TAO.Methods: This study consisted of two groups: the experimental group(TAO,11 cases)and the control group(Control,8 cases).The collection of posterior lens orbital connective tissue specimens was performed in 11 patientswith hyperthyroidism undergoing orbital decompression surgery.Eight patients with eyeball atrophy and eye socket atrophy due to eye trauma and other factors were selected as the control group.These patients had no active inflammation.The primary culture source for the orbital fibroblasts was selected after the eyeball was selected.According to the pre-experiment results,appropriate lentiviruses containing negative control group(Negative control,NC),miR-146 a mimic(miR-146am),miR-146a-5p-inhibition(miR-146ai)were added to the experiment.Individual wells of the group and the control group.Fluorescence microscopy was used to detect the transfection efficiency and morphology of infected orbital fibroblasts.CCK-8 was used to detect the optical density(OD)of 24 h,48 h,72 h,and 96 h,and cell survival was calculated.Rate(Cell viability).The orbital fibroblasts cultured in vitro were obtained by primary cell culture technique.After purification and culture,the laryngeal fibroblasts were transfected with the lentivirus carrying the gene of interest,and the expression level of miR-146 a in the orbital fibroblasts was overexpressed or inhibited.The orbital fibroblasts were successfully stained by laser scanning confocal microscopy and flow cytometry to analyze the localization and expression of IGF-1R receptors on the surface of orbital fibroblasts.Western blot(WB)was used to detect miR-146 a pairs.Effect of Akt protein expression in orbital fibroblasts after transfection.Finally,the expression level of TNF-? in the supernatant of orbital fibroblasts was detected by ELISA for 48 hours.Results: Successfully cultured primary orbital fibroblasts of connective tissue after human eyeballs in vitro,and successfully transfected lentivirus carrying miR-146 mimic(miR-146a)or miR-146 a inhibitor(miR-146ai)gene into eyelids In fibroblasts.After transfection,the infection efficiency of orbital fibroblasts was above 80%,and there was no significant change in themorphology of orbital fibroblasts.CCK-8 results showed that exogenous miR-146 mimic(miR-146am)promoted the proliferation of orbital fibroblasts,while miR-146 a inhibitor(miR-146ai)significantly inhibited the proliferation of orbital fibroblasts(P < 0.05)).Exogenous miR-146 am decreased the expression of IGF-1R on the surface of ocular fibroblasts in thyroid-associated ophthalmopathy,and miR-146 ai increased the surface of IGF-1R on the surface of orbital fibroblasts(P < 0.05).WB results suggested that exogenous miRNA-146 am reducesd the expression of Akt protein in orbital fibroblasts in thyroid gland(P <0.05).The level of TNF-? in the supernatant of orbital fibroblasts was significantly increased in TAO patients.The results were statistically different from the normal group(P<0.05).In the orbital fibrofines of patients with TAO,the level of TNF-? in the miR-146 am group decreased(P<0.05).At the same time,the level of miR-146 ai group increased(P<0.05).In normal human orbital fibroblasts,the level of TNF-a in miR-146 ai group was increased(P<0.05),and the level of TNF-a in miR-146 am group was not significantly different from that in Control-NC(P>0.05).Conclusion: Exogenous miR-146 am had no significant effect on the morphological changes of orbital fibroblasts,and significantly promoted the proliferation of orbital fibroblasts.miR-146 am inhibits the expression of IGF-1R and Akt on the surface of orbital fibroblasts in thyroid-associated ophthalmopathy,and miR-146 ai promotes the expression of IGF-1R and Akt in orbital fibroblasts.This effect suggests that IGF-1R/Akt may be an important pathway for TAO development,and exogenous miR-146 am may influence the development of TAO through the IGF-1R/Akt pathway.IGF-1R is expressed in TAO orbital fibroblasts,which are signaling molecules of orbital fibroblasts,and Akt molecules,as important downstream molecules in the known IGF-1Rsignaling pathway,may lead to hyaluronic acid or related Changes in inflammatory factors may be related to the development of TAO,which mediates the development of TAO.To study the effect of miR-146 a on IGF-1R/Akt in orbital fibroblasts may play a role in inhibiting the local cell proliferation and inflammatory response of TAO.Further research is needed.It is important to understand the pathogenesis of TAO and the development of specific treatment methods,which may become a new approach to TAO treatment.In addition,exogenous miR-146 am inhibited the expression of TNF-?in orbital fibroblasts,while miR-146 ai promoted the expression of TNF-? in orbital fibroblasts.miR-146 a has a positive effect on the inflammatory response.To study the role of miR-146 a in the proliferation of orbital fibroblasts,the expression of related signaling pathway proteins and the role of inflammatory response factors,is of great significance for understanding the pathogenesis of TAO and the development of specific therapeutic methods.