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Chidambaranathan Gowri Priya, Lavanya Kalaimani, Bharanidharan D, Venkatesh Prajna, Muthukkaruppan VR; Hsa-miR143-3p regulates human corneal epithelial stem cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4825.
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© ARVO (1962-2015); The Authors (2016-present)
Small RNA sequencing identified hsa-miR-143-3p to be highly expressed in enriched human corneal epithelial stem cells (CESCs-80%) compared to central corneal epithelial cells (CCECs). This study aims to understand the role of hsa-miR-143-3p in the regulation of CESCs.
The expression of hsa-miR-143-3p in enriched CESCs in comparison to CCECs was analysed by quantitative real time PCR (Q-RT-PCR) and by locked nucleic acid in-situ hybridization (LNA-ISH) in human corneo-limbal cryosections. Primary cultured limbal epithelial cells were transfected with has-miR-143-3p mimic, inhibitor and scrambled sequence (25 nM) using lipofectamine 3000. The transfected cells were analysed for (i) colony forming efficiency and (ii) expression of differentiation marker, several stem cell markers and the hsa-miR-143-3p predicted targets by Q-RT-PCR.
Q-RT-PCR analysis confirmed that the expression of hsa-miR-143-3p was higher (74±3.3-fold) in enriched CESCs compared to CCECs and was restricted to clusters of cells in limbal basal epithelium by LNA-ISH. Ectopic expression of miR-143-3p increased the colony forming efficiency (10.04 ± 0.45%) with the ability to form holoclones in comparison to inhibitor treated (0.27 ± 0.08%) and untreated cells (3.33 ± 0.23%). The mimic transfected cells showed higher expression of stem cell markers (ABCG2, NANOG, OCT4, KLF4 and ΔNP63) but reduced expression of differentiation marker (connexin-43). The hsa-miR-143-3p targets (KRAS, DVL 3, MAPK 1, MAPK 14) were downregulated thereby inhibiting Wnt and p38-MAPK pathways.
This is the first demonstration of the role of hsa-miR-143-3p in the maintenance of stemness in the human adult tissue resident stem cells.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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