Regulation of Ras homolog family member G by microRNA-124 regulates proliferation and migration of human retinal pigment epithelial cells

Jong Hwa Jun,Jae-Young Kim,Won-Ki Baek,Myeong-Jin Son,Hyun-Gyo Lee,Kyu Young Shim,Choun-Ki Joo

doi:10.1038/s41598-020-72360-5

Abstract

Uncontrolled retinal pigment epithelial (RPE) cell proliferation/migration contribute to the pathological tractional membrane development in proliferative vitreoretinopathy. Recent studies reported that microRNA (miR)-124 controls various cellular functions via the direct targeting of small Ras homolog family member G (RHOG). Therefore, we investigated the role of the neuron-specific miR-124 and RHOG in RPE cell proliferation/migration. Alterations in miR-124 and RhoG expression, as per cell confluence were evaluated through quantitative real-time PCR and western blotting, respectively. After transfection with miR-124, we quantified RPE cell viability and migration and observed cell polarization and lamellipodia protrusions. We evaluated the expression of RHOG/RAC1 pathway molecules in miR-124-transfected RPE cells. Endogenous miR-124 expression increased proportionally to RPE cell density, but decreased after 100% confluence. Overexpression of miR-124 decreased cell viability and migration, BrdU incorporation, and Ki-67 expression. Inhibition of endogenous miR-124 expression promoted RPE cell migration. Transfection with miR-124 reduced cell polarization, lamellipodia protrusion, and RHOG mRNA 3′ untranslated region luciferase activity. Like miR-124 overexpression, RhoG knockdown decreased RPE cell viability, wound healing, and migration, and altered the expression of cell cycle regulators. These results suggest that miR-124 could be a therapeutic target to alleviate fibrovascular proliferation in retinal diseases by regulating RPE proliferation/migration via RHOG.

Highlights

Physiology presents unique challenges that necessitate precisely tuned regulatory networks to maintain cell viability
Emerging evidence suggests that finetuning by miRNAs is essential for responses to cellular stress[15]; miRNAs play a critical role in the survival and physiology of photoreceptor cells and the retinal pigment epithelium, making them putative therapeutic targets in these blinding diseases
Endogenous expression levels of miR‐124 and RhoG inversely correlated with RPE cell conflu‐ ence

Summary

Introduction

Physiology presents unique challenges that necessitate precisely tuned regulatory networks to maintain cell viability. In steady-state conditions, RPE cells remain in the quiescent stage of the cell c ycle[9,10]. RPE cells are vulnerable to insults caused by the aging process and environmental stressors; proliferative vitreoretinopathy[11,12], diabetic retinopathy[13], and idiopathic epiretinal m embranes[14] can be induced by uncontrolled proliferation and RPE cell migration into vitreous space. Franke et al.[16] confirmed that miR-124 regulates neuronal differentiation by targeting RHOG. We investigated the regulatory functions of miR-124 in RPE cells; we focused at cell proliferation and migration. We assessed the physiological alterations caused by RHOG gene silencing in RPE cells, using small interfering RNAs (siRNAs)

Methods

Results

Conclusion