April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Altered Expression of MicroRNA in the MerTK Deficient RPE Cells and Their Potential Regulation of the Cytoskeletal Proteins During Phagocytosis
Author Affiliations & Notes
  • L. Han
    Ophthalmology, Kentucky Lions Eye Ctr, Univ of Louisville, Louisville, Kentucky
  • Q. Lu
    Beijing School of Ophthalmology, Capital Medical Uni., Beijing, China
  • Y. Wei
    Beijing School of Ophthalmology, Capital Medical Uni., Beijing, China
  • F. Ye
    Ophthalmology, Kentucky Lions Eye Ctr, Univ of Louisville, Louisville, Kentucky
  • N. Wang
    Beijing School of Ophthalmology, Capital Medical Uni., Beijing, China
  • Q. Li
    Ophthalmology, Kentucky Lions Eye Ctr, Univ of Louisville, Louisville, Kentucky
  • Q. Lu
    Ophthalmology, Kentucky Lions Eye Ctr, Univ of Louisville, Louisville, Kentucky
  • Footnotes
    Commercial Relationships  L. Han, None; Q. Lu, None; Y. Wei, None; F. Ye, None; N. Wang, None; Q. Li, None; Q. Lu, None.
  • Footnotes
    Support  The research was supported by Research to Prevent Blindness, a core grant (NIH EY015636), RR018733 (Q. Li); RR017702 (Q. Lu) and NIH/NEI -EY018830 (Q. Lu).
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 1675. doi:
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      L. Han, Q. Lu, Y. Wei, F. Ye, N. Wang, Q. Li, Q. Lu; Altered Expression of MicroRNA in the MerTK Deficient RPE Cells and Their Potential Regulation of the Cytoskeletal Proteins During Phagocytosis. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1675.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : MerTK receptor tyrosine kinase regulates RPE phagocytosis, and its deficiency causes photoreceptor degeneration and visual loss. This study has been performed to investigate MerTK regulation of microRNAs in retinal pigmental epithelial (RPE) cells, which regulate expression of their target genes that potentially affect phagocytosis.

Methods: : Both WT and MerTK mutant mice were housed in a 12-hour light-dark cycle with lights turned on at 0700 hr (7 AM) and off at 1900 hr (7 PM). For analysis of miRNA expression profile, we prepared total RNA samples from RPE by mechanically dissociation of the RPE sheets from the enucleated eyecups isolated from WT and MerTK mutant mice at 0900 hr and 1900 hr. After confirmation of the RNA integrity, miRNAs were fluorescent labeled and hybridized to miRCURYTM Array (Exiqon). The array images were collected with the Axon GenePix 4000B microarray scanner and digitized by GenePix pro V6.0 (Axon Instruments). Real-time qPCR analysis of miRNA expression was performed using Mir-X miRNA first-strand synthesis and quantification kit (Clontech).

Results: : Compared with WT RPE cells in the morning, 7 miRNAs in the mutants were significantly up-regulated, whereas 10 were significantly down-regulated. With similar comparison, 15 miRNAs were significantly up-regulated and 11 were down regulated in the evening. To search for those that may participate in regulating cytoskeletal protein expression during phagocytosis, we examined the potential target genes that might participate in phagosome formation. There were 11 among those altered miRNAs, which potentially regulated cytoskeletal protein expression. Downregulation of those proteins have been confirmed by qPCR.

Conclusions: : MerTK regulates RPE phagocytosis, and its deficiency causes photoreceptor degeneration and visual loss. Loss of MerTK alters microRNA expression, which in turn affects downstream target gene expression.

Keywords: phagocytosis and killing • gene microarray • retina 
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