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Qinghai Peng, Walter Collette, Michelle Twamley, Wenhu Huang, Annette John-Baptiste, Allison Vitsky; Ex vivo evaluation of miR-205 as a sensitive biomarker of corneal injury. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4679.
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© ARVO (1962-2015); The Authors (2016-present)
To examine levels of cornea-enriched microRNA-205 in ex vivo corneal culture following exposure to known corneal toxicant Tamoxifen, using lactate dehydrogenase (LDH), a non-specific cytotoxic indicator, as a comparator.
Corneas were harvested from 6 Long-Evans rats. Each cornea was dissected into 2 equal pieces, then washed and incubated in a 12-well plate with 2mL culture media. Culture media was refreshed ready for drug treatment following initial overnight incubation. Tamoxifen was added to wells at concentrations of 0.2µg, 2 µg and 20µg/ml. 200µl supernatant of culture media was collected at 3 and 24 hours post exposure for extraction of miRNA by Qiagen miRNeasy kit and the miR-205 was assayed via SYBR® Green-based qRT-PCR. Relative copy numbers were obtained utilizing known miR-205 oligos amplified by the same qRT-PCR as the reference. Data are presented as fold change relative to vehicle control ± SEM. LDH was assayed in parallel with these samples utilizing a Siemens Advia 1200.
When corneal explants were exposed to Tamoxifen, miR-205 levels within the culture media demonstrated both dose- and time-dependent increases. Compared to vehicle controls, corneas exposed to 20 µg/mL Tamoxifen demonstrated miR-205 elevations of ~9 fold at 3 hours and ~40 fold at 24 hours. In our model, miR-205 exhibited earlier and greater increases than LDH, which demonstrated ~4 fold elevation compared to vehicle-treated controls at the same dose and timepoint.
This study demonstrated earlier and greater increases of miR-205 levels than LDH post treatment with Tamoxifen in ex vivo culture, indicating that it could be a more sensitive novel biomarker in detecting corneal injury. Although miR-205 needs additional validation as a cornea specific biomarker of injury, these results provide promise for future studies such as in combination with other retinal miRNAs and additional corneal toxicants.
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