Abstract
Purpose :
The Ciliary Epithelium (CE) of adult mammalian eyes contains a quiescent population of retinal progenitor/stem cells that are able to proliferate, generate neurospheres in vitro and generate functional retinal neurons after induction. Despite this regenerative potential, the efficiency is still low, probably due to the presence of unknown mechanisms and inhibitory factors such as microRNAs (miRNAs). It is known that small non-coding RNAs are responsible to regulate a variety of progenitor and pluripotent gene expression, for that reason, we evaluate the effect of Let-7 family of miRNAs and its important target HMGA2 in CE cells during neurospheres formation
Methods :
CE cells from newborn Wistar rats (NB) were separated from retina, sclera and iris. The Pigmented Epithelium (PE) cells were cultured with growth factors (FGF and EGF, 10 and 20 ng/ml) to form neurospheres, together with chemically modified single-stranded oligonucleotides designed to bind and inhibit endogenous Let-7 miRNAs, or to mimic endogenous mature Let-7 (Let-7b, Let-7c and Let-7e) at early neurospheres formation stage. After 7 days in culture, we analyzed neurospheres size and number and expression of HMGA2 by PCR and Immunohistochemistry
Results :
NB PE cells highly expressed HMGA2 protein and transcripts, and decreased expression in adult tissues (200 folds decreased). Neurospheres obtained from NB PE cells expressed higher levels of HMGA2 than NB cells (33% higher). The opposite results were observed from Let-7 expression, where higher expression was observed in adults than in NB and Neurospheres cells. Experimentally increased expression of Let-7 did not change HMGA2 mRNA expression in neurospheres. However, Let-7 inhibition increased mRNA expression of HMGA2 (25%) and the number of neurospheres sized 20-50 µm (15% increased)
Conclusions :
Our results suggest that downregulation of Let-7 miRNAs increased HMGA2 expression and the number of small neurospheres, probably trough the regulation of the cell cycle. However, upregulation of Let-7 alone is not capable to regulate HMGA2. The information gleaned from this study may provide valuable insight into the cellular and molecular events that underlie the reprogramming response of CE cells and the mechanism of retinal recovery.
This is a 2020 ARVO Annual Meeting abstract.