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.

Identification of osteogenic differentiation-related miRNA-mRNA regulatory network of human periodontal ligament stem cells

“…In addition, the miR-143-3p inhibited osteogenic differentiation in dental pulp stem cells by binding to TNF-α via inactivating the NF-κB signaling pathway [21]. Interestingly, the miR-143-3p facilitated self-renewal and enhanced the expression of pluripotency genes in mouse embryonic stem cells, which revealed that hsa-miR-143-3p could maintain the stemness in corneal epithelial stem cells (CESCs) [22]. MiR-143-3p also suppressed glycolysis mentalism and reduced tumor formation via targeting HK2 [23].…”

Systems genetics identifies miRNA-mediated regulation of host response in COVID-19

“…Secondly, some of the miRNAs highlighted in our study are strong candidates for functional follow-up. For instance, hsa-miR-143-3p—which affects neutrophil counts mediated by BCL2 expression—has been implicated in numerous immunological diseases such as cancers and ischemic stroke [ 48 - 50 ], has been found to influence inflammatory factors and cell apoptosis [ 51 , 52 ], and to inhibit Wnt and MAPK signaling [ 53 ]. Similarly, hsa-miR-199b, which influences neutrophils through ETS1 expression, has previously been implicated in breast cancer [ 54 , 55 ], and described as a tumor suppressor in acute myeloid leukemia [ 56 ] and an inducer of apoptosis in oral cancer [ 51 , 57 ].…”

Mitochondrial impairment and downregulation of Drp1 phosphorylation underlie the antiproliferative and proapoptotic effects of alantolactone on oral squamous cell carcinoma cells

“…The cells were inoculated into a 6-well plate and transfected with control and Drp1 overexpressed plasmids. All transfections were performed using Lipofectamine 3000 (L3000008, Invitrogen, USA) according to the manufacturer’s instructions [ 30 ]. Transfection efficiency was confirmed by western blot.…”