title: rs78378222 polymorphism in the 3'-untranslated region of TP53 contributes to development of age-associated cataracts by modifying microRNA-125b-induced apoptosis of lens epithelial cells.
abstract: MicroRNAs (miRNAs) negatively regulate the expression of the target genes by binding to 'seed sequences' in the 3'‑untranslated region (3'‑UTR) mRNA transcripts, and the variants within or nearby 'seed sequences' may compromise or enhance miRNA/mRNA interaction leading to either 'loss‑of‑function' or 'gain‑of‑function' effects. Cataracts are the leading cause of blindness worldwide and are characterized by progressive aggregation and precipitation of lens proteins, and the development of age‑related cataracts is associated with dysregulated cellular activities of lens epithelial cells. Luciferase assays and online miRNA databases were used to validate that tumor protein p53 (TP53) is the target gene of miR‑125b. Furthermore, reverse transcription‑quantitative polymerase chain reaction and western blotting were conducted to detect expression levels of miR‑125b and TP53 in different groups of cells transfected with miR‑125b mimics or inhibitors. In addition, flow cytometry analysis and the MTT assay were conducted to detect the effects of miR‑125b on apoptosis and cell viability. The current study demonstrated that the rs78378222 polymorphism minor allele introduces a novel potential miR‑125b binding site in the TP53 3'‑UTR with a consecutive 8‑bp perfect match, creating a 'gain‑of‑function' variant and affecting the regulation of TP53 expression. A luciferase assay demonstrated that transfection of lens epithelial cells with wild type TP53 3'‑UTR significantly reduced the luciferase activity of the miR‑125b overexpressing cells compared with scramble controls. In addition, the luciferase activity of miR‑125b overexpressing cells transfected with the construct containing the rs78378222 polymorphism minor allele was also reduced compared with cells transfected with the wild type 3'‑UTR. Furthermore, it was demonstrated that the expression level of miR‑125 was comparable in epithelial cells from patients with age‑associated cataracts and controls, whereas the expression level of TP53 was significantly higher in the cataract group compared with controls. Additionally, transfection with 50 nM miR‑125b mimics markedly reduced the mRNA and protein expression levels of TP53 in the cultured lens epithelial cells, and miR‑125b significantly induced apoptosis in the epithelial cells compared with negative control cells. In conclusion, TP53 was identified as a target of miR‑125b, and the minor allele of the rs78378222 polymorphism promoted the miR-125b/TP53 mRNA interaction.
authors.affiliation: Beijing Tongren Eye Center, Beijing Key Laboratory of Ophthalmology and Visual Science, Beijing Tongren Hospital, Capital Medical University, Beijing 100073, P.R. China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Beijing Tongren Eye Center, Beijing Key Laboratory of Ophthalmology and Visual Science, Beijing Tongren Hospital, Capital Medical University, Beijing 100073, P.R. China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Li
authors.affiliation: Beijing Tongren Eye Center, Beijing Key Laboratory of Ophthalmology and Visual Science, Beijing Tongren Hospital, Capital Medical University, Beijing 100073, P.R. China.
authors.forename: Siquan
authors.initials: S
authors.lastname: Zhu
mesh_terms: D020413:3' Untranslated Regions; D000375:Aging / Q000235:genetics; D000483:Alleles; D017209:Apoptosis / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D002386:Cataract / Q000209:etiology*; D004847:Epithelial Cells / Q000378:metabolism; D015870:Gene Expression; D056726:Genetic Association Studies; D020022:Genetic Predisposition to Disease; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D034622:RNA Interference; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
chemical_list: D020413:3' Untranslated Regions; C531888:MIRN125 microRNA, human; D035683:MicroRNAs; D016159:Tumor Suppressor Protein p53