Abstract
BackgroundThis study is aimed to screen out the microRNAs (miRNAs) associated with H2O2 induced oxidative stress in human lens epithelial B3 (HLE-B3) cell lines and investigate their relations with the progression of age-related nuclear cataract.MethodsH2O2 was used to induce oxidative stress in HLE-B3 cells. A genome-wide expression profiling of miRNAs in HLE-B3 cells was performed to select the differentially expressed miRNAs before and after H2O2 treatment. The selected miRNAs were validated by RT-PCR and fluorescence in situ hybridization (FISH). Clinical specimens were divided into three groups according to the Lens Opacities Classification System III (LOCSIII) and the expression levels of the selected miRNAs were tested by RT-PCR in the three groups. Bioinformatics analyses were applied to predict the target genes of the miRNA hits and construct the miRNA regulatory network. The expression level of MAPK14 was analyzed by Western blot.ResultsThe H2O2 induced oxidative stress model of HLE-B3 cells was established. Nineteen upregulated and 30 downregulated miRNAs were identified as differentially expressed miRNAs. Seven of the total 49 were validated in the cell model. RT-PCR of the clinical samples showed that the expression levels of miR-34a-5p, miR-630 and miR-335-3p were closely related with the severity of nuclear opacity. The images taken from FISH confirmed the results of RT-PCR. There were 172 target genes of the three miRNAs clustered in the category of response to stress. The regulatory network demonstrated that 23 target genes were co-regulated by multiple miRNAs. MAPK14 was the target gene of three miRNAs and the result were verified by Western blot.ConclusionUp-regulation of miR-34a-5p and miR-630 and down-regulation of miR-335-3p are related with the progression of age-related nuclear cataract and the underlying mechanism awaits further functional research to reveal.
Highlights
This study is aimed to screen out the microRNAs associated with H2O2 induced oxidative stress in human lens epithelial B3 (HLE-B3) cell lines and investigate their relations with the progression of age-related nuclear cataract
H2O2 treatment decreased the viability of Human lens epithelial cells (HLEs)-B3 cells In the current study, we used H2O2 to generate excessive reactive oxygen species (ROS), which can permeate cellular membranes and enter into the cells to cause oxidative damage
The viability of HLE-B3 cells exposed to various concentrations of H2O2 after 24 h incubation was investigated by MTT assay
Summary
This study is aimed to screen out the microRNAs (miRNAs) associated with H2O2 induced oxidative stress in human lens epithelial B3 (HLE-B3) cell lines and investigate their relations with the progression of age-related nuclear cataract. Human lenses are transparent in young people, but changes occur as the body ages These changes include the development of a hard, compact nucleus, local opacity, and, the development of a pathological cataract [1]. Many factors such as diabetes mellitus, ultraviolet, systemic drugs and congenital diseases are known to be related to cataract formation Among these factors, oxidative stress with the generation of reactive oxygen species (ROS) is thought to be a major predisposing factor in age-related cataracts [2]. There has no record of a systemic screening for oxidative stress associated miRNAs in human lens epithelial cells (HLECs)
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