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S. J. Spurr-Michaud, I. K. Gipson; Membrane-Associated Mucins MUC1 and MUC16 Differ in Barrier Function at the Ocular Surface. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1946.
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MUC1 and MUC16 are major membrane-associated mucins present in the glycocalyx of the apical surface of human corneal epithelium. We have previously shown that MUC16 functions as a component of the barrier on human corneal epithelial cells that prevents rose bengal dye penetrance. The purpose of this study was to investigate whether MUC1 has a similar function.
Immortalized human corneal limbal epithelial cells (HCLE) were stably transfected with siRNA specific to MUC1 using the plasmid psiRNA driven by the H1 promoter (InvivoGen), similar to that done for MUC16 (Blalock et al., 2007). Cells were then grown for optimal mucin expression (7 days post confluence in serum containing medium). Expression of MUC1, 4 and 16 mRNA were determined by qPCR and MUC1 protein by western blot in transfected cells and compared to non-transfected and vector transfected controls. Barrier function was assessed in MUC1 knockdown, non-transfected and vector transfected cultures with the anionic dye rose bengal. Areas taking up the dye (evidence of lack of protection) were quantitated using ImageJ software. Statistical significance was determined by the Mann-Whitney test in Instat3 statistical software.
MUC1 mRNA and protein were knocked down by 60% (p<0.05) and 66% (p<0.05), respectively, in the stably transfected HCLEsiMUC1 cells. Message for MUC4 and MUC16 were not significantly altered. There was a significant increase in rose bengal dye exclusion in the HCLEsiMUC1 cells compared to non-transfected (p<0.0001) and vector transfected (p<0.001) controls (N=4), indicating that MUC1 is not functioning to prevent dye penetrance.
MUC1 does not recapitulate the barrier role of MUC16 in human corneal epithelial cells as measured by rose bengal dye penetrance. The increase in barrier function with MUC1 knockdown could be a result of a more homogeneous MUC16 rich glycocalyx.
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