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Jhansi Rani kasinathan, Veerappan Muthukkaruppan, Chidambaranathan Gowri Priya, Corneal Epithelial Stem Cells; miR-203 inhibits ΔNp63α dependent clonogenicity in corneal epithelial stem cells (CESCs). Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4899.
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© ARVO (1962-2015); The Authors (2016-present)
One of the isoforms of the nuclear transcription factor p63 - ΔNp63α is expressed higher in CESCs in comparision to the differentiated cells. In keratinocytes, miR-203 has been reported to repress stemness by inhibiting ΔNp63α expression. This study aims to elucidate whether miR-203 has a similar influence in ΔNp63α isoform expression and in the maintanence of stemness in the non-keratinized corneal epithelium.
Limbal explant cultures were carried out using human globes (Arpitha et al., 2008). After 21 days of culture, the cells were trypsinized and 1.5 X 105cells were transfected with 20nm pre-miR-203, antago-miR-203 and scrambled sequence using HiPerfect transfection reagent (Qiagen) (Lena et al., 2008). After 48 hours of transfection, (i) 2000 cells were seeded in mitomycin treated 3T3 feeder layer and stained for rhodamine after culturing for 7 days to evaluate their colony forming efficiency (Arpitha et al., 2008). (ii) RNA extraction was performed using Qiagen RNeasy mini kit followed by semi quantitative RT-PCR to analyze the expression of ΔNp63α and β isoforms (Di Iorio et al., 2005) in the transfected cells.
Semi quantitative RT-PCR analysis revealed that pre-miR-203 represses ΔNp63α expression compared to control (transfected with scrambled sequence). In contrast, antago-miR-203 enhanced ΔNp63α expression. Similarly, colony forming efficiency of cells treated with antago-miR-203 was higher (2.2%) compared to control (1.4%) and was significantly reduced to 0.1% with pre-miR-203.
miR-203 specifically inhibits the proliferative potential in CESCs by regulating ΔNp63α expression. Further studies are essential to understand the signaling pathways associated with this molecular regulation of stemness.
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