LncRNA LPAL2/miR-1287-5p/EGFR Axis Modulates TED-Derived Orbital Fibroblast Activation Through Cell Adhesion Factors.

CONTEXT: The activation of orbital fibroblasts, the prime targets in thyroid eye disease (TED), is central to its underlying pathogenesis. OBJECTIVE: We aimed to investigate the mechanism of TED orbital fibroblast activation from the perspective of noncoding RNA regulation. METHODS: Immunofluorescence (IF) staining was applied to evaluate the fibrotic changes in target cells. Cell proliferation was evaluated by 5-ethoxy 2-deoxyuridine and colony-formation assays. Collagen I concentration was determined by enzyme-linked immunosorbent assay. Human microarray analysis was performed on 3 TED and 3 healthy control orbital tissue samples. RESULTS: Bioinformatics analysis showed that cell adhesion signaling factors were differentially expressed in TED tissues, including intercellular adhesion molecule (ICAM)-1, ICAM-4, vascular cell adhesion molecule, and CD44, which were all upregulated in diseased orbital tissues. Long noncoding RNA LPAL2 level was also upregulated in orbital tissues and positively correlated with ICAM-1 and ICAM-4 expression. Stimulation of the TED orbital fibroblasts by transforming growth factor-ß1 (TGF-ß1) significantly increased the expression of ICAM-1, ICAM-4, and LPAL2. Knockdown of LPAL2 in orbital fibroblasts inhibited TGF-ß1-induced increases in cell adhesion factor levels and orbital fibroblast activation. Microarray profiling was performed on TED and normal orbital tissues to identify differentially expressed microRNAs, and miR-1287-5p was remarkably reduced within diseased orbital samples. miR-1287-5p was directly bound to the epidermal growth factor receptor (EGFR) 3' untranslated region and LPAL2, and LPAL2 modulated EGFR/protein kinase B (AKT) signaling through targeting miR-1287-5p. CONCLUSION: The LPAL2/miR-1287-5p axis modulated TGF-ß1-induced increases in cell adhesion factor levels and TED orbital fibroblast activation through EGFR/AKT signaling.

The Smad2/3/4 complex binds miR-139 promoter to modulate TGFß-induced proliferation and activation of human Tenon's capsule fibroblasts through the Wnt pathway.

The activation and proliferation of human Tenon's fibroblasts (HTFs) play a vital role in the fibrosis in the pathology of the scar formation after the glaucoma filtration surgery. Transforming growth factor ß1 (TGFß1)/Smads signaling has been reported to promote fibrosis. In our previous study, we revealed that TGFß1-induced orbital fibroblast activation and proliferation through Wnt/ß-catenin signaling. As microRNA (miR)-139 could target several factors in Wnt signaling to modulate fibrosis, here, the effect and mechanism of miR-139 in HTF activation and proliferation were investigated. miR-139 overexpression significantly reversed the TGFß1-induced increase in collagen I and α-smooth muscle actin contents and proliferation in HTFs. CTNNB1 and CTNND1 were direct downstream of miR-139 and can significantly restore the suppressive effect of miR-139 on the activation and proliferation in HTFs under TGFß1 stimulation. Smad2/3/4 complex inhibits the transcription activity of miR-139, most possibly by Smad4 binding to the miR-139 promoter. Taken together, we demonstrated a new mechanism of HTF activation and proliferation from the perspective of miRNA regulation, which may provide new strategies for improving the fibrosis after the glaucoma filtration surgery.

Low density lipoprotein - rosiglitazone - chitosan-calcium alginate/nanoparticles inhibition of human tenon's fibroblasts activation and proliferation.

Anti-fibrotic therapeutic methods with safety and efficiency after glaucoma filtration surgery (GFS) are desirable. In our previous study, by using Human Tenon's Fibroblasts (HTFs) as a model, we proved the expression of peroxisome proliferator activates receptor-γ (PPAR-γ) in HTFs; in addition, rosiglitazone (RSG), an agonist of PPAR-γ, can inhibit transforming growth factorsß1 (TGF-ß1)-induced reactivation of HTFs, thus to inhibit specifically scarring after GFS through intervening TGF-ß/Smads signal pathway. However, a better drug delivery way of RSG, to prolong the duration of its function, and to reduce the toxicity of RSG to ocular tissue still remains challenges. Low density lipoprotein receptor (LDLr) is strongly expressed in hyper-proliferation HTFs after GFS. Therefore, we structured targeting LDL-RSG complexes and channel them into HTFs through LDL-LDLr pathway in order to promote anti-proliferation of HTFs and reduce the toxicity to ocular tissue. Meanwhile, in order to improve the release properties of LDL-RSG complexes, we structured slow release system of LDL-RSG/chitosan-calcium alginate - nanoparticles (CSNP), which effectively inhibited TGF-ß1-induced HTFs proliferation, synthesis of extracellular matrix and activation of TGF-ß1/SMAD pathway. These data suggested that LDL-RSG/CSNP can be a new anti-fibrotic therapeutic method on scarring after GFS and also a novelty administration of RSG.

MiRNA-21 promotes fibrosis in orbital fibroblasts from thyroid-associated ophthalmopathy.

PURPOSE: This study aimed to determine the role of miR-21 in orbital fibroblasts obtained from donors with thyroid-associated ophthalmopathy (TAO) and to elucidate the regulation of fibrosis by miR-21 in the pathological process of TAO. METHODS: The expression of miR-21 was investigated in orbital tissues from 26 donors with TAO and 10 donors without TAO. Human orbital fibroblasts were cultivated from TAO donors, and the role of miR-21 in orbital fibroblast proliferation, apoptosis, and differentiation was analyzed. Moreover, the effect of transforming growth factor-beta1 (TGF-ß1) on miR-21 expression was also analyzed. In addition, the regulation of miR-21 in TGF-ß1-induced collagen production was determined. RESULTS: The expression of miR-21 in orbital fibroblasts from TAO was higher than in donors without TAO. Additional experiments demonstrated that miR-21 enhanced proliferation, decreased apoptosis, and promoted differentiation in TAO orbital fibroblasts. Moreover, this study also showed that TGF-ß1 induced miR-21 expression in a time- and dose-dependent manner and miR-21 promoted collagen I mRNA expression and total collagen production induced by TGF-ß1. Additionally, miR-21 activated the TGF-ß1/Smad signaling pathway by enhancing Smad3 phosphorylation. CONCLUSIONS: The present study shows that miR-21 regulates cell proliferation, apoptosis, and differentiation in orbital fibroblasts from TAO, and acts as a mediator in TGF-ß1-induced collagen production. These data predict a close association between miR-21 and orbital muscle fibrosis, and provide a novel therapeutic target for TAO.

MicroRNA-29 mediates TGFß1-induced extracellular matrix synthesis by targeting wnt/ß-catenin pathway in human orbital fibroblasts.

PURPOSE: Transforming growth factor ß1 (TGFß1) is very important in the synthesis and degradation of extracellular matrix (ECM) and also in the mediation of human orbital fibroblasts (OFs) proliferation. MicroRNA-29 (MiR-29) plays an important role in this process. In the present study, the effects of TGFß1 on the expression of miR-29 and whether miR-29 is involved in pro-survival signaling pathways mediated by TGFß1 were examined in human OFs. METHODS: Detecting the influence of TGFß1 on the expression of miR-29a/b/c by real-time PCR analysis. Using 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) to detecting the influence of miR-29 on the increased proliferation caused by TGF-ß1 on the human orbital fibroblasts. Using soft agar assay to detecting the influence of miR-29 on the increased colony formation caused by TGF-ß1 on the human orbital fibroblasts. Western blot was used to detect the specific mechanisin. RESULTS: TGFß1 treatment decreases the expression of miR-29 in OFs. In the cultured OFs, the value of optical density (OD) in the group treated with miR-29 is lower than that in the group treated without miR-29 (P < 0.05). In the cultured OFs, the ratio of colony formation in the group treated with miR-29 is lower than that in the group treated without miR-29 (P < 0.05). In OFs, miR-29 decreases the secretion of Wnt3a and activation of ß-catenin whether the treatment of TGFß1 was used or not. MiR-29 decreases expression of Collagen, type I, alpha 1 (COL1A1) through down-regulation of wnt/ß-catenin pathway. CONCLUSIONS: In OFs TGFß1 treatment decreases expression of miR-29 which can cause the inhibition of normal ability of TGFß1. MiR-29 inhibits TGFß1-induced proliferation of OFS cell and decreases colony formation of OFS cell after TGFß1 treatment. MiR-29 Mediates TGFß1-induced Extracellular matrix synthesis through activation of Wnt/ß-catenin pathway in human OFs.