miRNAs in the vitreous humor of patients affected by idiopathic epiretinal membrane and macular hole

Abstract

Purpose: The aim of the present study was to assess the expression of miRNAs in the Vitreous Humor (VH) of patients with Macular Hole (MH) and Epiretinal Membrane (ERM) compared to a control group.

Methods: In this prospective, comparative study, 2-ml of VH was extracted from the core of the vitreous chamber in consecutive patients who underwent standard vitrectomy for ERM and MH. RNA was extracted and TaqMan® Low Density Arrays (TLDAs) were used to profile the transcriptome of 754 miRNAs. Results were validated by single TaqMan® assays. Finally, we created a biological network of differentially expressed miRNA targets and their nearest neighbors.

Results: Overall 10 eyes with MH, 16 eyes with idiopathic ERM and 6 controls were enrolled in the study. Profiling data identified 5 miRNAs differentially expressed in patients affected by MH and ERM with respect to controls. Four were downregulated (miR-19b, miR-24, miR-155, miR-451) and 1 was downregulated (miR-29a); TaqMan® assays of the VH of patients affected by MH and ERM, with respect to controls, showed that the most differentially expressed were miR-19b (FC -9.13, p:<0.00004), mir-24 (FC -7.52, p:<0.004) and miR-142-3p (FC -5.32, p:<0.011). Our network data showed that deregulation of differentially expressed miRNAs induces an alteration of several pathways associated with genes involved in both MH and ERM.

Conclusion: The present study suggests that disregulation of miR-19b, miR-24 and miR-142-3p, might be related to the alterations that characterize patients affected by MH and ERM.

Fig 1. Box Plots of differentially expressed miRNAs.

Box Plots from Single TaqMan^® Assays on TaqMan^® Low Density Arrays (TLDA) of differentially expressed miRNAs. Validation by single TaqMan^® assays of differentially expressed miRNAs identified by TLDAs in the vitreous humor of all the patients and controls. Values on the y-axis are reported as ΔCt × (−1).

Fig 2. Biological processes controlled by miR-19b, miR-24 and miR-142-3p network.

Overrepresented biological functions from a molecular network built on validated targets of differentially expressed miRNAs, retrieved from different annotation databases (GO, KEGG, Reactome). On the left of each histogram the overrepresented pathways are reported, while on the right the corresponding associated genes to MHs and ERMs based on literature data are shown. Data are plotted as—log10 of p-values for each biological process.

Fig 3. Receiver Operator Characteristic (ROC) curves for vitreal miR-19b, miR-24 and miR-142-3p in patients affected by MHs and ERMs.

ROC curves of miR-19b (A), miR-24 (B), miR-142-3p (C) DCts in comparison with ERMs + MHs vs controls; miR-19b (D), miR-24 (E), miR-142-3p (F) DCts in comparison with ERMs vs controls; miR-19b (D), miR-24 (E) in comparison with MHs vs controls. Curves represent DCts calculated by using U6 as endogenous control.