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Mariam Lotfy Khaled, Zhong Chen, Mitchell A Watsky, Yutao Liu; Molecular characterization of MIR184 mutation in keratoconus using primary human corneal cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1403.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Keratoconus (KC) is the most common cornea ectasia. Mutations in MIR184 have been identified in KC patients; however, the exact mechanism remains unclear. miR-184 regulates cellular proliferation either by directly inhibiting AKT2 expression or indirectly via protecting INPPL-1 from degradation. We aimed to study the molecular impacts of mutated miR-184 on the expression of AKT2 and INPPL-1 in primary human corneal epithelial cells (HCEC) and stromal fibroblast cells (SFC).
Method: Primary HCEC and SFC were isolated from human donor eyes. Cells were cultured in 6-well plates and transfected with 50nM of miR-184 mimic, mutated miR-184 (r. 57C>U), miR-184 inhibitor, or negative control. After 24 hours culture, both RNA and protein were collected from the cells for further analysis. The expression of miR-184, AKT2 and INPPL-1 levels in HCEC and SFC was measured using droplet digital PCR (ddPCR). The house-keeping gene PRLP0 was used as the reference gene for normalization. A proliferation assay was performed in triplicate for human corneal epithelial cells after transfection and the fluorescence intensity was measured 1, 3, and 6 days after transfection.
Results: ddPCR confirmed the expression alterations of miR-184 in HCEC and SFC with the corresponding transfections. Compared to the negative controls, HCEC and SFC transfected with the miR-184 mimic showed 25% and 40% reduction in AKT2 expression, respectively (p <0.01). Both the mutant and the miR-184 inhibitor showed no inhibition in HCEC or SFC AKT2 levels. INPPL-1 expression in the HCEC or SFC cells was not significantly different as compared to any of the miR-184 transfections. HCEC transfected with miR-184 mutant or miR-184 inhibitor showed significantly increased cell proliferation (p<0.01) as compared to miR-184 mimic transfected cells. Western blot analysis is being conducted to examine protein expression alterations.
Conclusion: Mutation in MIR184 decreases its functional efficiency in regulating corneal epithelial proliferation. Mutated miR-184 leads to the dysregulation of AKT2 expression in both corneal epithelial and stromal cells. Deregulation of the AKT pathway may play a role in corneal abnormalities showed in KC.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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