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Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-β Signaling
Published on: August 3, 2018
Interfering Hsa_circRNA_0060640 Suppresses TGF-β2-Induced Proliferation, Motility and EMT in Human Lens Epithelium
Ming Guo1, Fanfan Su1, Yao Chen1
1Department of Ophthalmology, Jingzhou Hospital, Yangtze University (Jingzhou Central Hospital), Jingzhou, China.
Background:
Circular RNA (circRNA) is a novel star factor in the research of ocular diseases including cataract and the most common postoperative complication posterior capsule opacification (PCO). Hsa_circRNA_0060640 (circ_0060640) is an age-related cataract-related circRNA. However, its role in cataractogenesis is unrevealed yet.
Methods:
PCO in vitro model was established in human lens epithelium cells (hLECs) induced by transforming growth factor-beta2 (TGF-β2). RNA and protein expressions were respectively detected by quantitative PCR and western blotting. Direct interaction between two RNAs was predicted by Starbase tool and confirmed by dual-luciferase reporter assay. MTS and EdU assays measured cell proliferation; Transwell, starch wound and western blotting assays evaluated cell motility and epithelial-mesenchymal transition (EMT).
Results:
Circ_0060640 expression is higher in anterior lens capsule tissues from human cataractous eyes and TGF-β2-stimulated hLECs cells line SRA01/04. RNA interference of circ_0060640 could prevent SRA01/04 cells from TGF-β2-induced cell proliferation, migration and invasion, accompanied with decreased N-cadherin and α-smooth muscle actin and increased E-cadherin. Mechanistically, circ_0060640 directly controls microRNA (miR)-214-3p expression and then regulates gene expression of collagen type I alpha2 chain (COL1A2). Notably, COL1A2 inhibition is underlying the protective role of circ_0060640 silencing and miR-214-3p ectopic expression in TGF-β2-stimulated SRA01/04 cells.
Conclusion:
Circ_0060640 is a novel cataract-related gene and its silencing could block TGF-β2-evoked hLECs proliferation, motility and EMT in vitro via targeting miR-214-3p-COL1A2 axis. Therefore, targeting circ_0060640 via RNA interference might be a treatment strategy for PCO development.
Insights
Circular RNA (circRNA) hsa_circRNA_0060640 is elevated in cataractogenesis and drives posterior capsule opacification (PCO) by promoting cell proliferation and migration. Silencing circ_0060640 may offer a therapeutic strategy for PCO.
Area of Science:
- Ophthalmology
- Molecular Biology
- Genetics
Background:
- Circular RNAs (circRNAs) are emerging factors in ocular diseases like cataract and posterior capsule opacification (PCO).
- Hsa_circRNA_0060640 (circ_0060640) is identified as an age-related cataract-associated circRNA, but its specific role remains unclear.
- Posterior capsule opacification (PCO) is a common complication following cataract surgery.
Purpose of the Study:
- To investigate the role of hsa_circRNA_0060640 in cataractogenesis and PCO.
- To elucidate the underlying molecular mechanisms of circ_0060640 in human lens epithelial cells (hLECs).
Main Methods:
- Established an in vitro PCO model using TGF-β2-induced hLECs.
- Quantified circ_0060640 expression via qPCR and protein expression via Western blotting.
- Assessed cell proliferation (MTS, EdU), motility (Transwell, wound healing), and epithelial-mesenchymal transition (EMT) markers.
- Confirmed direct RNA-RNA interaction using dual-luciferase reporter assay.
Main Results:
- Circ_0060640 expression was significantly upregulated in cataractous human lens tissues and TGF-β2-treated hLECs.
- Silencing circ_0060640 inhibited TGF-β2-induced hLEC proliferation, migration, and EMT.
- Circ_0060640 directly targets miR-214-3p, subsequently regulating COL1A2 expression.
- COL1A2 inhibition mediated the protective effects of circ_0060640 silencing and miR-214-3p restoration.
Conclusions:
- Circ_0060640 acts as a novel cataract-related gene promoting hLEC proliferation, motility, and EMT via the miR-214-3p-COL1A2 axis.
- Targeting circ_0060640 through RNA interference presents a potential therapeutic strategy for preventing PCO development.
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