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Evelyn Png, S S. Yong, H L. Yeo, E Mendoz, M C. Leong, C T. Lim, L Tong; Transglutaminase is Important for Cell Adhesion and Migration in Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):284.
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© ARVO (1962-2015); The Authors (2016-present)
Transglutaminase (TGM)-2 is a multi-functional cross linking protein that performs important roles in the ocular surface. In previous studies, we showed in human corneal epithelial cells that TGM-2 regulated the rate of wound closure during in-vitro scratch test assays since RNA interference against TGM-2 reduced the speed of wound closure. We aimed to study the underlying mechanism of this observation by evaluating the effect of TGM-2 on cell adhesion and migration.
We use cultured human corneal epithelial cells that expressed a short hairpin RNA against TGM-2 (shTG) and scrambled sequence shRNA (control). A non-invasive cell impedance assay (Xcelligence) was used to evaluate cell adhesion properties up to 7 hours after seeding. Cells were treated with low concentration of trypsin and detachment was recorded with live microscopy to evaluate adhesion strength. Image analysis of single cells was performed to evaluate spreading pattern. The proliferation of cells after scratching was performed using Ki-67 staining and cell cycle analysis was performed by mini-flow cytometry.
The morphology of shTG and control cells was similar when grown on normal and fibronectin-coated plates. The impedance of shTG cells was greatly reduced up to 7 hours after seeding compared to control. Control cells, compard to shTG required a significantly longer period of time before cell detachment after trypsinisation. Both shTG and control cells demonstrated the similar areas of spreading at 5 hours. Once attached, these cells were extending lamellopodia in various directions visualised. The proportion of proliferating (Ki-67 positive) cells was not significantly different between the control cells or shTG cells at 6 hours after scratching. Cell cycling was not different between shTG and control.
The TGM-2 status of human corneal epithelial cells controls cell adherence rather than cell proliferation. TGM-2 targeting or replacement may have therapeutic relevance in human ocular surface diseases involving wound healing processes.
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