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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)
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.
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.
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.
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.
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