טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentNagossa Sara
SubjectRole of MicroRNA-184 in the Skin and Corneal Pathophysiology
DepartmentDepartment of Medicine
Supervisor Professor Ruby Shalom-Feuerste


Abstract

Adult stem cells (SC) which are found throughout the body regenerate the tissue in which they reside, under homeostasis or stress/injury. Despite much advance in our understanding of the biology of SCs, the key mechanisms that control early exit from stemness state, including the cues that control SC self-renewal, differentiation and proliferation, are partially understood.

In this study, we focused on the skin (epidermis) and corneal epithelium which serve as excellent models for SC research. The dynamic replenishment of these tissues depends on very similar epithelial SCs. Elucidating the molecular circuitry of skin and corneal SCs and their regulation is required for understanding the etiology of skin/corneal disease and developing new therapies for numerous epithelial diseases.

MicroRNAs play a role in various physiological processes including SC regulation. MiR-184 is a highly evolutionary conserved gene and its mutations lead to corneal/lens blinding disease, underscoring the importance of this gene. Recently, it was shown that while miR-184 is not expressed by epidermal/corneal SCs, it is highly expressed by early committed cells, suggesting that it may induce SC differentiation. However, miR-184-related function remained unclear partially due to the lack of in vivo model.

In this thesis, we characterized a new model, Mir184-null mouse, and Mir184 transgenic mouse to force miR-184 expression by SCs using “tet-on” system. Mir184-null mice displayed an increase in the expression of the stem/progenitor marker p63, and epidermal hyperplasia linked with increased cell proliferation. In agreement, Mir184 transgenic mouse reduced proliferation, induced epidermal thinning and differentiation. In vitro study has demonstrated that miR-184 activated Notch pathway and differentiation through direct repression of two stem/progenitor cell factors, namely, K15 and FIH1. Similar to the depletion of Notch1 in mice, Mir184-null mice developed more skin tumors as compared to wild-type mice. These experiments indicated that miR184/Notch1 axis induced early exit from stemness state and behaved as tumor suppressors in the skin.

In accordance, Mir184-null corneas showed abnormal commitment of limbal SC (LSC) which coincided with enhanced cell proliferation of committed progenitors. In line, Mir184-deficient mice displayed delayed wound closure after corneal injury. Similar to the phenotype of some patients carriers of MIR184 mutation, Mir184-knockout mice displayed corneal stromal thinning. RNA sequencing and in silico analysis revealed a set of genes that were controlled by miR-184 and are linked with cell adhesion, response to injury, epithelial-stromal crosstalk and keratoconus. Additionally, occasional detachment of the corneal epithelium from the underlying stroma was evident in tissue sections of Mir184-null cornea that were correlated with a drastic reduction in the basement-membrane protein Nidogen-1 and its upstream regulator, the transcription factor Myb.

Altogether, we propose that by activating Notch1, miR-184 induces a commitment switch from stemness to differentiation and serves as an epidermal tumor suppressor. MiR-184 regulates epithelial-stromal interactions to control corneal stromal thickness, and adhesion to the basement-membrane. These findings suggest that miR-184 is an essential regulator of SC differentiation and the new models developed here may serve as a platform for drug discovery for corneal pathologies.