September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Autophagy, a protective factor in limbal epithelium, is positively regulated by a microRNA family
Author Affiliations & Notes
  • Han Peng
    Northwestern University, Chicago, Illinois, United States
  • Jong Kook
    Northwestern University, Chicago, Illinois, United States
  • Julia Katsnelson
    Rush Medical College, Chicago, Illinois, United States
  • Wending Yang
    Northwestern University, Chicago, Illinois, United States
  • CongCong He
    Northwestern University, Chicago, Illinois, United States
  • Robert M Lavker
    Northwestern University, Chicago, Illinois, United States
  • Footnotes
    Commercial Relationships   Han Peng, None; Jong Kook, None; Julia Katsnelson, None; Wending Yang, None; CongCong He, None; Robert Lavker, None
  • Footnotes
    Support  National Institutes of Health Grants EY06769, EY017539 and EY019463 (to R.M.L.); DK094980 (to C.C.H); a Dermatology Foundation research grant and Career Development Award (to H.P); and a MidWest Eye Bank research grant (H.P)
Investigative Ophthalmology & Visual Science September 2016, Vol.57, No Pagination Specified. doi:
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      Han Peng, Jong Kook, Julia Katsnelson, Wending Yang, CongCong He, Robert M Lavker; Autophagy, a protective factor in limbal epithelium, is positively regulated by a microRNA family. Invest. Ophthalmol. Vis. Sci. 201657(12):.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose : Autophagy plays critical roles in maintenance of tissue homeostasis and is an important protective factor in tissues governed by stem cells. The autophagic process in corneal epithelial stem cells is under studied

Methods : We used antagomirs (antagos) to knock-down miR-103, -107 or -124 (irrelevant-antago) in cultured human limbal epithelial keratinocytes (HLEKs) and a LC3-GFP mouse. To characterize the vacuoles, we used contrast light, immunofluorescence, and transmission electron microscopy. To determine the origin of the vacuoles, pharmacological and genetic inhibition at different stages of autophagy was performed. Bioinformatic analysis combined with luciferase assays and biochemistry identified autophagy-related miRNA targets

Results : In HLEKs, antagos-103/107 cause the formation of large hybrid vacuoles, which are positive for lysosomal (LAMP1 and lysotrackor), autophagosomal (LC3), and macropinosomal (Rabankyrin-5) markers. Such vacuoles were rescued by inhibiting autophagosomal-lysosomal fusion, indicative that vacuole accumulation occurred after autophagolysosome formation and is due to a failure of end-stage autophagy. An increase in LC3II and p62 was noted indicating an attenuation of autophagy flux by antago treatment. More LC3-GFP puncta (a measure of autophagy) were observed in limbal (miRs-103/107-enriched) versus corneal (miRs-103/107-low) basal epithelia, indicative of a high degree of autophagy in the stem cell-enriched limbal epithelium. Active autophagy in this compartment is consistent with autophagy being a stem cell protective factor. In support of this idea, a decrease of holoclone (stem cell-derived) colonies, was detected in HLEKs with compromised autophagic capability. Dynamin, an essential protein for hybrid vacuoles recycling in end stage autophagy, is phosphorylated (inactive) by PKC and p35. Phosphatidylcholine-specific phospholipase D 1/2 and sphingomyelin synthase 2, upstream activators of PKC signaling, were shown to be direct targets of miRs-103/107, as was p35. Inhibition of PKC and p35 pathways decreased p-dynamin and rescued the accumulation of hybrid vacuoles

Conclusions : miRs-103/107 are critical for regulating proliferation, proliferative capacity and cell-cell communication in the limbal epithelium. The addition of autophagy to the regulatory portfolio of miRs-103/107 highlights the importance of this family in stem cell biology

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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