Abstract
Purpose:
Autophagy degrades unnecessary cellular components, often stress-related. Most attention has been paid to early-stage autophagy; however, little is known about end stages, which include clearing autophagolysosomes and regenerating lysosomes. Little attention has been paid to autophagy in the corneal/limbal epithelium.
Methods:
Using a LC3-GFP mouse, the degree of autophagy in corneal and limbal epithelia was evaluated. We used antagomirs (antagos) to knock-down miR-103, -107 or -124 (irrelevant-antago). To characterize the vacuoles, we used contrast light, immunofluorescence, and transmission electron microscopy. To determine the origin of the vacuoles, pharmacological inhibition of different stages of autophagy was performed. mRNA profiling, combined with bioinformatic analysis, luciferase assays and biochemistry identified miRNA targets related to autophagy.
Results:
More LC3-GFP punctas (a measure of autophagy) were observed in limbal basal versus corneal basal epithelia, indicative of a higher activity of autophagy in the stem cell-enriched limbal epithelium. This is correlated with higher levels of miRs-103/107 in the limbal versus corneal epithelium supporting the idea that this miRNA family is involved in autophagy. Consistent with this idea, loss of miRs-103/107 in primary human limbal epithelial keratinocytes (HLEKs) resulted in the formation of numerous vacuoles devoid of organellar material. Such vacuoles were rescued by: (i) pharmacologically inhibiting the initiation of autophagy, suggesting an autophagic origin of these vacuoles; and (ii) blocking autophagosomal-lysosomal fusion, indicating that the vacuoles were induced after autophagolysosome formation. Thus, accumulation of vacuoles following loss of miRs-103/107 is due to failure of end-stage autophagolysosome clearance. mRNA profiling revealed that treatment of HLEKs with antagos-103/107, upregulated FOXO3 and CREB5, which are predicted targets in silico. Luciferase assays and biochemistry confirmed that FOXO3 and CREB5, genes related to the orderly clearance of autophagic vacuoles, were bona fide targets.
Conclusions:
miRs-103/107 have been reported to be critical for regulating proliferation, proliferative capacity and cell-cell communication in the limbal epithelium. The addition of autophagy, a process vital for stem cell maintenance, to the regulatory portfolio of miRs-103/107 highlights the importance of this family in stem cell biology.