MicroRNAs are important components of mechanisms that regulate normal and pathogenic biological processes. Glaucoma, which is the leading cause of blindness worldwide, is a complex disease associated with increased intraocular pressure (IOP) caused by restricted aqueous humor drainage through the trabecular meshwork (TM). In this study, we aimed to identify candidate miRNAs with effects relevant to glaucoma and the TM.

Two web sites devoted to miRNAs, MiRwalk and miR2Disease, and available literature were used to identify microRNAs potentially involved in glaucoma and TM functions. Among miRNAs identified, one that was selected for further studies was submitted to MiRWalk to predict its target genes. DisGeNET, STRING and KEGG bioinformatics tools were used to prioritize the target genes. To empirically verify the predictions, the 3'UTR of selected genes was cloned downstream of a luciferase reporter gene in a psiCHECK2 vector. The effects of the miRNA were assessed by dual luciferase assays after transfections in Hek-293 and TM. Ultimately, effects on endogenous TM proteins were tested by real time PCR and Western blotting experiments.

Twenty one miRNAs potentially involved in glaucoma and TM functions were identified. Mir-204 was selected for further study because it has been shown to be involved in various eye functions. Among its target genes, twelve were selected to be best candidates for relevance to glaucoma and the TM. Dual luciferase assays confirmed that mr-204 down regulated seven of the genes. Down regulation of the endogenous RNAs and proteins of five of these genes in TM cells by mir-204 was shown, respectively, by real time PCR and Western blot analysis.

Several miRNAs potentially involved in glaucoma related functions, including mir-204, were predicted using bioinformatics tools. Among multiple predicted targets of mir-204, twelve best candidates for relevance to glaucoma were identified and the effects of five of these in primary TM cells were empirically shown. Three of the five genes have known apoptosis related functions. Apoptosis is thought to be involved in the etiology of glaucoma, and abnormal functioning of these genes may thus contribute to the glaucoma phenotype.