Unraveling linc00645: A Wnt-regulated lncRNA driving epithelial-mesenchymal transition through the linc00645/miR-452-5p/vimentin axis in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with a poor prognosis and high recurrence rates. Wnt signaling plays a critical role in the pathogenesis of TNBC, with aberrant activation of this pathway observed in many cases. Emerging evidence suggests that long non-coding RNAs (lncRNAs) may modulate key signaling pathways, including Wnt signaling, in TNBC. In this study, we investigated the interplay between lncRNAs and Wnt pathway activation in TNBC. RNA-Seq analysis comparing Wnt-positive and Wnt-negative cell lines derived from MDA-MB-231 identified linc00645 as a highly upregulated lncRNA in Wnt-activated cells. Further gene ontology analysis revealed that the differentially expressed lncRNAs, including linc00645, were enriched in pathways related to epithelial-to-mesenchymal transition (EMT), a key process in cancer metastasis. To validate these findings, recombinant human Wnt3A was used to activate the Wnt pathway in MDA-MB-231 cells. RT-qPCR confirmed the upregulation of linc00645 in Wnt-activated cells. Functional studies demonstrated that knockdown of linc00645 led to a significant reduction in cell proliferation and migration in TNBC cells, suggesting its oncogenic role. Moreover, mechanistic investigations revealed that linc00645 directly interacts with miR-452-5p, leading to the downregulation of this microRNA and the upregulation of the mesenchymal marker vimentin, further promoting EMT. These findings suggest that linc00645 functions as an oncogenic lncRNA in Wnt-activated TNBC by regulating the linc00645/miR-452-5p/vimentin signaling axis. Targeting this axis could represent a novel therapeutic strategy for treating TNBC. This study highlights the potential of linc00645 as a promising target for interventions aimed at inhibiting TNBC progression. Overall design: The 7TGP vector, which has an EGFP encoding sequence downstream of an inducible promoter with seven Tcf repeats (7xTcf-eGFP) 27 was stably transfected into low passage MDA-MB-231 cells. Fluorescence-activated cell sorting (FACS) was then employed to enrich for EGFP-positive (EGFP+) or EGFP-negative (EGFP-) cells from the MDA-MB-231 cells stably transfected with the 7TGP vector. The expanded EGFP+ and EGFP- cells were then again subjected to FACS. After eight rounds of expansion followed by FACS, the resultant MDA-MB-2317TGP-POS cell line was greater than 80% EGFP+, while the MDA-MB-2317TGP-NEG cell line was greater than 95% EGFP-.