The objective of the study was to elucidate the microRNA (miRNA) profile of an enriched human corneal epithelial stem cell (CESC) population in comparison to differentiated central corneal epithelial cells (CCECs) by small RNA sequencing. The CESCs were enriched by differential enzymatic treatment to isolate the basal limbal epithelial cells followed by laser capture microdissection of cells with nucleus to cytoplasm ratio ≥0.7, from donor tissues. Small RNA sequencing was carried out using Illumina NextSeq. 500 platform and the validation of differentially expressed miRNAs by quantitative real-time PCR (qPCR) and locked nucleic acid miRNA in-situ hybridization (LNA-ISH). The sequencing identified 62 miRNAs in CESCs and 611 in CCECs. Six miRNAs: hsa-miR-21-5p, 3168, 143-3p, 10a-5p, 150-5p and 1910-5p were found to be significantly upregulated in enriched CESCs, which was further confirmed by qPCR and LNA-ISH. The expression of hsa-miR-143-3p was exclusive to clusters of limbal basal epithelial cells. The targets of the upregulated miRNAs were predicted to be associated with signaling pathways -Wnt, PI3K-AKT, MAPK and pathways that regulate pluripotency of stem cells, cell migration, growth and proliferation. Further studies are essential to elucidate their functional role in maintenance of stemness. The findings of the study also hypothesize the inherent potential of hsa-miR-143-3p to serve as a biomarker for identifying CESCs.
Our previous study demonstrated hsa-miR-143-3p as one of the highly expressed miRNAs in enriched corneal epithelial stem cells (CESCs). Hence this study aims to elucidate the regulatory role of hsa-miR-143-3p in the maintenance of stemness in CESCs. The target genes of hsa-miR-143-3p were predicted and subjected to pathway analysis to select the targets for functional studies. Primary cultured limbal epithelial cells were transfected with hsa-miR-143-3p mimic, inhibitor or scrambled sequence using Lipofectamine 3000. The transfected cells were analysed for (i) colony forming potential, (ii) expression of stem cell (SC) markers/ transcription factors (ABCG2, NANOG, OCT4, KLF4, ΔNp63), (iii) differentiation marker (Cx43), (iv) predicted five targets of hsa-miR-143-3p (DVL3, MAPK1, MAPK14, KRAS and KAT6A), (v) MAPK signaling regulators and (vi) Wnt-β-catenin signaling regulators by qPCR, immunofluorescence staining and/or Western blotting. High expression of hsa-miR-143-3p increased the colony forming potential (10.04 ± 1.35%, p < 0.001) with the ability to form holoclone-like colonies in comparison to control (3.33 ± 0.71%). The mimic treated cells had increased expression of SC markers but reduced expression of Cx43 and hsa-miR-143-3p targets involved in Wnt-β-catenin and MAPK signaling pathways. The expression of β-catenin, active β-catenin and ERK2 in hsa-miR-143-3p inhibitor transfected cells were higher than the control cells and the localized nuclear expression indicated the activation of Wnt and MAPK signaling. Thus, the probable association of hsa-miR-143-3p in the maintenance of CESCs through inhibition of Wnt and MAPK signaling pathways was thus indicated.
Purpose The 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). Methods Small 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. Results The 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. Conclusion A regulatory role for hsa‐miR‐150‐5p in maintenance of stemness by inhibiting Wnt signaling pathway is thus indicated.
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