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Hsa-miR-143-3p inhibits Wnt-β-catenin and MAPK signaling in human corneal epithelial stem cells.

ABSTRACT: 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.

SUBMITTER: Kalaimani L

PROVIDER: S-EPMC9259643 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The aim of the present study was to identify the differentially expressed microRNAs (DEMs) between Lynch syndrome (LS) and the normal colonic (N-C) control samples, predict the target genes (TGs) and analyze the potential functions of the DEMs and TGs. The miRNA expression dataset GSE30454, which included data of 13 LS and 20 N-C tissue samples, was downloaded from the Gene Expression Omnibus. The classical t-test in Linear Models for Microarray Data package was used for DEM identification. TG prediction was performed using 5 databases. The regulatory network of the DEMs and their TGs was constructed using Cytoscape. Functional and pathway enrichment analysis was performed. The transcription factors (TFs), tumor-associated genes (TAG) and tumor suppressor genes (TSGs) were then identified. Three key DEMs hsa-miR-137, hsa-miR-520e, and hsa-miR-590-3p were identified. Hsa-miR-520e and hsa-miR-137 had 4 common TGs, including SNF related kinase, metal-regulatory transcription factor 1 (MTF1), round spermatid basic protein 1 and YTH N6-methyladenosine RNA binding protein 3; hsa-miR-590-3p and hsa-miR-137 had 14 common TGs, including NCK adaptor protein 1 (NCK1), EPH receptor A7, and stress-associated endoplasmic reticulum protein 1; hsa-miR-590-3p and hsa-miR-520e had 12 common TGs, including Krüppel-like factor (KLF) 13, twinfilin actin binding protein 1, and nuclear factor I B. Through the functional and pathway enrichments analysis, MTF1 was involved in regulation of gene expression and metabolic processes, and sequence-specific DNA binding TF activity. KLF13 was involved in regulation of gene expression and regulation of cellular metabolic processes. NCK1 was enriched in the axon guidance pathway. In addition, the functional and pathway enrichment analysis showed certain TGs, such as hypoxia-inducible factor 1α, AKT serine/threonine kinase 2, and rapamycin-insensitive companion of mammalian target of rapamycin, participated in the mTOR signaling pathway. The 3 key DEMs hsa-miR-137, hsa-miR-520e, and hsa-miR-590-3p may have important roles in the process of LS.

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Hsa-miR-143-3p inhibits Wnt-β-catenin and MAPK signaling in human corneal epithelial stem cells.

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