miR-199b-5p directly regulates the expression of ZEB1 and Snail1 in corneal endothelial cells

Abstract

Purpose : Widespread microRNA (miRNA) expression is downregulated in Fuchs endothelial corneal dystrophy (FECD) and we have found miRNA sequences to be hypermethylated in FECD (Khuc et al. PLoS One 2017). Specifically, miR-199b-5p expression is almost completely silenced and its promoter is significantly hypermethylated. The purpose of this study was to investigate how altered miR-199b expression may contribute to FECD pathogenesis.

Methods : The relationship between miR-199b-5p and ZEB1 and Snai1 was evaluated by bioinformatics analysis and dual-luciferase reporter gene assays. Putative miR-199b-5p target genes were predicted using different computational algorithms including miRmap (http://mirmap.ezlab.org/) and TargetScan (http://www.targetscan.org/vert_71/). Fragments of both ZEB1 and SNAI1 3’UTRs with wild-type and mutated binding sites were cloned into the luciferase reporter vector and tested in a human corneal endothelial cell line (HCEnC-21T).

Results : Putative miR-199b-5p target genes prediction scores ranged from 50-100. ZEB1 and Snai1 were selected for further analysis based on their high scores in both prediction tools (83.4 and 97). In silico analysis revealed that the 3’ UTR of human ZEB1 (positions 1023-1029; NM_001128128.2) and Snai1 (positions 725-731; NM_ 005985.3) mRNAs contain a highly conserved binding site for miR-199b-5p. Dual-luciferase reporter assays revealed that miR-199b-5p directly binds the 3’UTR of ZEB1 and Snai1. miR-199b-5p mimic suppressed the luciferase activities of the pMIR-ZEB1-WT (~30%, p= 0.009) and pMIR-SNAI1-WT (~50%, p=0.039) compared with the scrambled negative control. By contrast, miR-199b-5p inhibitor blocked the suppressive effect (p<0.05). miR-199b-5p mimic and inhibitors had no effect on luciferase activities of pMIR-ZEB1-Mut, pMIR-SNAI1-Mut, or empty luciferase vector without any insert DNA.

Conclusions : FECD pathogenesis involves the abnormal deposition of extracellular matrix (ECM). ZEB1 and Snai1 overexpression in corneal endothelial cells leads to excessive ECM deposition in FECD. Our results show that miR-199b-5p directly binds to and negatively regulates ZEB1 and Snai1 expression. Thus, hypermethylation of miR-199b-5p found in FECD may result in abnormally increased expression of ECM proteins by corneal endothelial cells.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.