miRNA-195-5p modulates cell proliferation and stemness by targeting the Wnt signalling network in breast cancer

Abstract

Cancer stem cells (CSCs) are a specific subpopulation of cells within the tumour characterised by self-renewal, proliferation and tumorigenic potential. Heterogeneity in the CSCs population and their ability to rewire the signalling networks make it difficult to target cancer by single gene inhibition. This study investigates the regulatory role of miR-195-5p in modulating CSC properties and associated signalling pathways in breast cancer. Previously shown to inhibit proliferation, invasion, and metastasis, miR-195-5p is now revealed to downregulate stemness markers (OCT4, SOX2, NANOG, and CD44). The prevalence of heterogeneity of stemness markers in MCF-7 and MDA-MB-231 cell lines is a novel advancement of this study. In MCF-7, SOX2-and CD44-overexpressing cells were found to be distinct, non-overlapping populations, as confirmed by immunofluorescence colocalisation analysis. SOX2-positive nuclei comprised ∼30 % of the MCF-7 population and were significantly reduced following miR-195-5p overexpression. Mechanistically, this downregulation of stemness correlated with decreased expression of β-catenin and its upstream regulators GSK3β and FZD6, were validated as direct targets of miR-195-5p using a luciferase reporter assay. miR-195-5p also inhibited TCF/LEF transcriptional activity, indicating suppression of canonical Wnt/β-catenin signalling. Functionally, miR-195-5p overexpression led to a reduced proliferation zone in CFSE-prestained 3D spheres derived from MCF-7 cells over seven days. To further dissect its mechanism, Wnt signalling was perturbed using siRNA against GSK3β, β-catenin and ICG-001 (a CBP/β-catenin interaction inhibitor), and their combination. GSK3β knockdown led to increased β-catenin expression, nuclear localisation, and enhanced 3D sphere proliferation, while β-catenin inhibition upregulated protein expression of GSK3β and suppressed proliferation of cancer cells. Combined inhibition of GSK3β and CBP/β-catenin interaction downregulated expression of β-catenin, supporting the role of miR-195-5p in CSC suppression. This study identifies miR-195-5p as a potent regulator of CSCs and proliferation, and modulator of the Wnt signalling cascade. Co-inhibition of GSK3β and CBP/β-catenin through miR-195-5p highlights its therapeutic potential in combating stemness and proliferation in breast cancer.