Screening And Verification Of Oxidation-related MicroRNAs And Target Genes In Human Lens Epithelial Cells And The Research On The Mechanism Of MiR-34a-5p Regulating Oxidative Damage

Objective: 1.To find out and verify the oxidation-related miRNAs and their target genes in HLE-B3 cells.2.To investigate the mechanism of miR-34a-5p and its target genes in regulating oxidative damage on HLE-B3 cell line.3.To provide new targets for the research on the pathogenesis of age-related nuclear cataract.Methods: 1.H₂O₂ was applied to introduce oxidative damage on HLE-B3 cells and the effect was monitored by MTT,flowcytometry and Hoechst staining;2.Microarray was used to screen out the differentially expressed miRNAs and the result was validated by q RT-PCR and FISH;3.Bioinformatic and proteomics analysis were performed to pick the target genes for the miRNA and their regulatory relationship were verified externally by dual-luciferase reporter gene assay and internally by q RT-PCR and Western blot;4.Transfection was facilitated to manipulate the expression level of miRNA and its target genes and the viability of HLE-B3 cells were detected by CCK8 assay.Results:1.After incubation with 75?M H₂O₂ for 24 h,the viability of HLE-B3 cells were decreased to 76.22±2.64%.The percentage of apoptosis cells was 41.5%,which was detected by flow cytometry and Hoechst staining.2.After induction of oxidative danmage on HLE-B3 cells,49 differentially expressed miRNAs were discovered by microarray.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.3.Sixty-eight target genes associated with stress?oxidative stress included?were picked from over 1000 total target genes by target prediction combined with proteomic and bioinformatic analysis.SIRT1 and GPX3 were chosen as the final candidates.Endogenous verification found that the RNA and protein expression of SIRT1 and GPX3 were decreased by overexpression of miR-34a-5p,while the results were reversed by inhibition of miR-34a-5p.Dual luciferase assay showed that miR-34a-5p transfection could down-regulate the relative luciferase activity ratio of wild type plasmid containing 3'-UTR region of SIRT1 and GPX3,but had no effect on that of mutant and NC plasmid in which the sequences of 3'-UTR region of SIRT1 and GPX3 were unidentifiable for miR-34a-5p.4.Upon oxidative damage,the RNA and protein expression of SIRT1 and GPX3 were declined.This effect,along with the recession of cell viability,was exaggerated by up-regulation of miR-34a-5p,while down-regulation of miR-34a-5p could compensate the downfall.Conclusions: MiR-34a-5p is a key element in the regulation of oxidative damage on HLE-B3 cells and the underlying mechanism may be its negative regulation on SIRT1 and GPX3,which results in reduced cell viability and anti-oxidative ability.