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Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-β Signaling
Published on: August 3, 2018
Transforming Growth Factor-β1-induced Human Subconjunctival Fibrosis is Mediated by MicroRNA 143/145 Expression
Young Hoon Hwang1, Sun-Ah Jung1, Jungmook Lyu2
1Myung-Gok Eye Research Institute, Kim's Eye Hospital, Konyang University College of Medicine, Seoul, Korea.
Purpose:
To investigate the roles and pathways of microRNAs 143 and 145 in transforming growth factor (TGF)-β1-induced human subconjunctival fibrosis.
Methods:
Human tenon's capsule fibroblasts (HTFs) were obtained from a healthy eye. After treating cultured HTFs with TGF-β1, the expression of microRNAs 143 and 145 was evaluated using polymerase chain reaction. To identify the pathways of TGF-β1-induced microRNA 143/145 expression, HTFs were treated with specific inhibitors of p38MAPK, PI3K/Akt, JNK, ERK, and with siRNAs for SMAD2 and SMAD4. Mutagenesis studies were performed to evaluate the role of the CArG box and SMAD-binding element (SBE). To investigate the role of microRNA 143/145 in TGF-β1-induced myofibroblast transdifferentiation, microRNA 143/145 mimics and microRNA 143/145 inhibitors were applied to the HTFs.
Results:
Array analysis revealed that TGF-β1 induced the expression of microRNA 143/145 in a dose- and time-dependent manner. When inhibitors and siRNAs for p38MAPK, PI3K/Akt, ERK, and JNK were applied, the TGF-β1-induced expression of microRNA 143/145 was inhibited; however, SMAD2 and SMAD4 inhibition did not affect the TGF-β1-induced expression of these microRNAs. In the mutagenesis studies, both the CArG box and SBE were associated with TGF-β1-induced expression of microRNA 143/145. Mimics of microRNA 143/145 induced increased myofibroblast formation, whereas their inhibitors had the opposite effect.
Conclusions:
TGF-β1-induced human subconjunctival fibrosis was mediated by the expression of microRNA 143/145, mainly via SMAD-independent pathways. Inhibition of TGF-β1-induced microRNA 143/145 expression in HTFs might represent a novel strategy to prevent subconjunctival fibrosis.
Insights
MicroRNAs 143 and 145 are key players in transforming growth factor-β1-induced subconjunctival fibrosis. Inhibiting these microRNAs in human tenon
Area of Science:
- Ocular surface disease
- Fibrosis research
- MicroRNA biology
Background:
- Subconjunctival fibrosis is a significant cause of ocular surface disease, often exacerbated by transforming growth factor-β1 (TGF-β1).
- MicroRNAs (miRNAs) are emerging as critical regulators in fibrotic processes.
- Understanding the specific roles of miRNAs, such as miR-143 and miR-145, in TGF-β1-mediated fibrosis is crucial for developing targeted therapies.
Purpose of the Study:
- To elucidate the roles and underlying pathways of microRNAs 143 and 145 in TGF-β1-induced human subconjunctival fibrosis.
- To investigate the molecular mechanisms by which TGF-β1 influences the expression of miR-143 and miR-145 in ocular fibroblasts.
Main Methods:
- Human tenon's capsule fibroblasts (HTFs) were cultured and treated with TGF-β1.
- Expression levels of miR-143 and miR-145 were quantified using polymerase chain reaction.
- Specific inhibitors and siRNAs targeting signaling pathways (p38MAPK, PI3K/Akt, JNK, ERK, SMAD2, SMAD4) and mutagenesis studies (CArG box, SBE) were employed to dissect regulatory mechanisms. MiRNA mimics and inhibitors were used to assess functional impact on myofibroblast differentiation.
Main Results:
- TGF-β1 significantly upregulated miR-143 and miR-145 expression in HTFs in a dose- and time-dependent manner.
- Inhibition of p38MAPK, PI3K/Akt, ERK, and JNK pathways attenuated TGF-β1-induced miR-143/145 expression, while SMAD2/4 inhibition had no significant effect.
- Both CArG box and SMAD-binding element (SBE) were implicated in TGF-β1-induced miRNA expression. MiR-143/145 mimics promoted myofibroblast formation, whereas inhibitors reduced it.
Conclusions:
- TGF-β1-induced subconjunctival fibrosis is mediated by miR-143 and miR-145 through predominantly SMAD-independent pathways.
- Targeting miR-143/145 expression represents a potential therapeutic strategy for preventing TGF-β1-driven subconjunctival fibrosis.
- This study highlights the critical role of specific microRNAs in the pathogenesis of ocular fibrotic conditions.
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