May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Effects of MUC16 Knockdown in Human Corneal-Limbal Epithelial Cells
Author Affiliations & Notes
  • T. D. Blalock
    Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts
  • A. S. Tisdale
    Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts
  • S. R. Heimer
    Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts
  • S. J. Spurr-Michaud
    Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts
  • M. S. Gilmore
    Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts
  • I. K. Gipson
    Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts
  • Footnotes
    Commercial Relationships T.D. Blalock, None; A.S. Tisdale, None; S.R. Heimer, None; S.J. Spurr-Michaud, None; M.S. Gilmore, None; I.K. Gipson, None.
  • Footnotes
    Support NIH F32 #EY016937 to TDB, NIH R01 #EY03306 to IKG, and NIH R01 #EY08289 to MSG.
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 474. doi:
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      T. D. Blalock, A. S. Tisdale, S. R. Heimer, S. J. Spurr-Michaud, M. S. Gilmore, I. K. Gipson; Effects of MUC16 Knockdown in Human Corneal-Limbal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):474.

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

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Abstract

Purpose:: MUC16 is a membrane-associated mucin present in the glycocalyx, particularly on the tips of the microplicae of the ocular surface epithelium. Little is known about the function of MUC16 on the ocular surface, thus the effects of MUC16 expression knockdown in human corneal-limbal epithelial cells were measured.

Methods:: Immortalized human corneal-limbal epithelial cells (HCLE) were stably transfected with two sequences of siRNA specific to MUC16 using retrovirus produced by 293-10A1 cells transfected with pSuperRetro (Oligoengine, Inc). Expression in transfected cells was measured by quantitative real time PCR and immunoblotting using antibodies to MUC16 (OC125) and was compared to non-transfected and vector-transfected controls. MUC16 knockdown cultures were incubated with the anionic dye rose bengal for 5 minutes to assess uptake as a measure of barrier function. The area of islands of stratified cells that excluded rose bengal was quantified in culture images using ImageJ analysis software. Also, MUC16 knockdown cells were incubated with fluorescently labeled Staphylococcus aureus followed by fixation with paraformaldehyde. Immunofluorescence microscopy was performed using antibodies to MUC1 (HMFG-2) or MUC16 (OC125) and nuclei were labeled with DAPI. The amount of adherent bacteria was quantified using ImageJ analysis software.

Results:: The more effective of the siRNA sequences reduced MUC16 mRNA by 65% and MUC16 protein by 90% with no significant effect on control genes (MUC1, MUC4, and GAPDH) and no noted effect on microplicae structure. Cells expressing this siRNA sequence showed an 82% decrease in areas of stratified cells that exclude rose bengal compared to controls. In addition, a 3.6-fold increase of labeled adherent S. aureus was observed in the MUC16 knockdown cultures compared to negative controls.

Conclusions:: A MUC16 knockdown cell line was created that showed significant reduction of rose bengal uptake and increased binding of S. aureus. These data suggest that MUC16 forms a protective barrier on the ocular surface.

Keywords: cornea: epithelium • cornea: surface mucins • cornea: tears/tear film/dry eye 
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