Hsa‐miR‐150‐5p regulates human corneal epithelial stem cells through Wnt signaling

Abstract

AbstractPurposeThe aim of this study was to understand the molecular regulatory function of hsa‐miR‐150‐5p in association with the maintenance of stemness in corneal epithelial stem cells (CESCs).MethodsSmall RNA sequencing identified hsa‐miR‐150‐5p to be highly expressed in enriched CESCs compared to central corneal epithelial cells (CCECs). The validation of differential expression of hsa‐miR‐150‐5p in enriched CESCs in comparison to CCECs was carried out by quantitative real time PCR (Q‐PCR) and by locked nucleic acid in‐situ hybridization (LNA‐ISH) in human corneo‐limbal cryosections. Primary cultured limbal epithelial cells were transfected with hsa‐miR‐150‐5p mimic, inhibitor and scrambled sequence (25 nM) using lipofectamine 3000. The transfected cells were analysed for (i) colony forming potential (ii) expression of stem cell markers (ABCG2, NANOG, OCT4, KLF4 and ΔNP63), differentiation marker (connexin‐43) and the hsa‐miR‐150‐5p predicted targets (JARID 2, AKT 3, INHBA and CTNNB1) by Q‐PCR and (iii) expression of ABCG2, p63, connexin‐43, JARID 2, AKT 3 and catenin by immunofluorescence staining.ResultsThe expression of hsa‐miR‐150‐5p was higher (13.86±1.5) in enriched CESCs compared to CCECs by Q‐PCR analysis. Hsa‐miR‐150‐5p had relatively higher expression in clusters of cells in limbal basal epithelium compared to other layers by LNA‐ISH. Ectopic expression of miR‐150‐5p increased the colony forming potential (8.28 ± 0.33%) with the ability to form holoclones in comparison to inhibitor treated (0.71 ± 0.10%) and control (1.8 ± 0.15%). The mimic treated cells had higher expression of stem cell markers but reduced expression of connexin‐43 and hsa‐miR‐150‐5p targets involved in Wnt signaling pathway.ConclusionA regulatory role for hsa‐miR‐150‐5p in maintenance of stemness by inhibiting Wnt signaling pathway is thus indicated.