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CircMAP3K4 protects human lens epithelial cells from H2O2-induced dysfunction by targeting miR-193a-3p/PLCD3 axis in
Yu Ma1, Yi Liu2, Baotong Shu3
1Department of Ophthalmology, Zhengzhou University, Affiliated Hospital 5, Zhengzhou, Henan, China.
Abstract:
Circular RNAs (circRNAs) have shown pivotal regulatory roles in multiple human ocular diseases, including age-related cataract (ARC). Here, we explored the role of circRNA mitogen-activated protein kinase kinase kinase 4 (circMAP3K4, hsa_circ_0078619) in ARC pathology and its associated mechanism. The expression of RNAs and proteins was examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot assay. Cell viability, senescence, proliferation, and apoptosis were analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, senescence-associated-β-galactosidase (SA-β-Gal) staining, 5-ethynyl-20-deoxyuridine (EdU) assay, and flow cytometry. The oxidative stress status of SRA01/04 cells was analyzed using the commercial kits. The interaction between microRNA-193a-3p (miR-193a-3p) and circMAP3K4 or phospholipase C delta 3 (PLCD3) was verified by dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay, and RNA-pull down assay. CircMAP3K4 was significantly down-regulated in ARC patients and H2O2-induced SRA01/04 cells. H2O2 treatment restrained the viability and proliferation and promoted the senescence, apoptosis, and oxidative stress of SRA01/04 cells, and circMAP3K4 overexpression protected SRA01/04 cells from H2O2-induced dysfunction. MiR-193a-3p was a direct target of circMAP3K4, and circMAP3K4 overexpression-mediated protective effects in H2O2-induced SRA01/04 cells were largely reversed by the accumulation of miR-193a-3p. MiR-193a-3p interacted with the 3' untranslated region (3'UTR) of PLCD3, and PLCD3 knockdown largely overturned miR-193a-3p silencing-induced protective effects in H2O2-induced SRA01/04 cells. CircMAP3K4 up-regulated the expression of PLCD3 via sponging miR-193a-3p in SRA01/04 cells. In conclusion, circMAP3K4 protected SRA01/04 cells from H2O2-induced dysfunction in ARC through mediating miR-193a-3p/PLCD3 axis.
Insights
Circular RNAs (circRNAs) like circMAP3K4 are downregulated in age-related cataract (ARC). Overexpressing circMAP3K4 protects ocular cells from oxidative stress by regulating the miR-193a-3p/PLCD3 pathway, offering therapeutic potential for ARC.
Area of Science:
- Ophthalmology
- Molecular Biology
- Genetics
Background:
- Circular RNAs (circRNAs) play critical roles in human ocular diseases, including age-related cataract (ARC).
- The specific role and mechanism of circMAP3K4 (hsa_circ_0078619) in ARC remain largely unexplored.
Purpose of the Study:
- To investigate the function of circMAP3K4 in age-related cataract (ARC) pathology.
- To elucidate the underlying molecular mechanism involving circMAP3K4, microRNA-193a-3p (miR-193a-3p), and phospholipase C delta 3 (PLCD3) in ocular cells.
Main Methods:
- Quantitative reverse transcription PCR (RT-qPCR) and Western blot to assess RNA and protein expression.
- Cell viability, senescence, proliferation, and apoptosis assays (MTT, SA-β-Gal, EdU, flow cytometry) to evaluate cellular function.
- Dual-luciferase reporter, RNA immunoprecipitation (RIP), and RNA-pull down assays to confirm molecular interactions.
Main Results:
- CircMAP3K4 expression was significantly decreased in ARC patients and hydrogen peroxide (H₂O₂)-induced ocular cells.
- H₂O₂-induced cellular dysfunction, including reduced viability/proliferation and increased senescence/apoptosis/oxidative stress, was ameliorated by circMAP3K4 overexpression.
- CircMAP3K4 directly targeted miR-193a-3p, which in turn targeted PLCD3; circMAP3K4 exerted protective effects by sponging miR-193a-3p and upregulating PLCD3.
Conclusions:
- CircMAP3K4 functions as a protective factor against oxidative stress-induced cellular damage in the context of age-related cataract.
- The circMAP3K4/miR-193a-3p/PLCD3 axis represents a novel regulatory pathway in ARC pathogenesis.
- CircMAP3K4 holds potential as a therapeutic target for age-related cataract.
