Identification of H2O2 induced oxidative stress associated microRNAs in HLE-B3 cells and their clinical relevance to the progression of age-related nuclear cataract

Abstract

Abstract Background 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. Methods H2O2 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. Results The 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. Conclusion Up-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.

Keywords

• Age-related nuclear cataract
• Oxidative stress
• microRNA
• Bioinformatics analysis