Abstract
mRNA-circRNA-miRNAs axes have been characterized in breast cancer, but not as risk-assessment axes for tumor initiation in early-onset breast cancer that is increasing drastically worldwide. To address this gap, we performed circular RNA (circRNA) microarrays and microRNA (miRNA) sequencing on acini of HMT-3522 S1 (S1) breast epithelial risk-progression culture model in 3D and chose an early-stage population miRNome for a validation cohort. Nontumorigenic S1 cells form fully polarized epithelium while pretumorigenic counterparts silenced for gap junction Cx43 (Cx43-KO-S1) lose epithelial polarity, multilayer and mimic premalignant in vivo mammary epithelial morphology. Here, 121 circRNAs and 65 miRNAs were significantly dysregulated in response to Cx43 silencing in cultured epithelia and 15 miRNAs from the patient cohort were involved in epithelial polarity disruption. Focusing on the possible sponging activity of the validated circRNAs to their target miRNAs, we found all miRNAs to be highly enriched in cancer-related pathways and cross-compared their dysregulation to actual miRNA datasets from the cultured epithelia and the patient validation cohort. We present the involvement of gap junction in post-transcriptional axes and reveal Cx43/hsa_circ_0077755/miR-182 as a potential biomarker signature axis for heightened-risk of breast cancer initiation, and that its dysregulation patterns might predict prognosis along breast cancer initiation and progression.
Highlights
Among key players involved in breast cancer onset are gap junction components
Silencing Cx43 expression in these nontumorigenic S1 cells via Cx43-shRNA (Cx43KO-S1) resulted in cell cycle entry, perturbed apical polarity, mitotic spindle misorientation and loss of lumen, causing cell m ultilayering[19] and priming cells for enhanced motility and invasion[19,20]. These phenotypic features observed in Cx43-KO-S1 acini represent architectural and phenotypical premalignant mammary lesions, like those observed in ductal hyperplasia in a murine m odel[23], which increase the risk of breast cancer initiation, marking Cx43-KO-S1 as a pretumorigenic culture model
While dysregulated mRNA-circular RNA (circRNA)-miRNAs axes have been reported as biomarker signatures in cancers[30,36,37,38] and in breast c ancers[39,40,41,42,43,44,45], axes involved in premalignant epithelial polarity transitions that might explain and contribute to heightened risk of breast cancer initiation have not been reported
Summary
Among key players involved in breast cancer onset are gap junction components. Gap junction intercellular communication (GJIC) is mediated by transmembrane proteins, called connexins (Cxs) that allow the intercellular exchange of ions, second messengers and metabolites between adjacent c ells[9,10,11,12]. Silencing Cx43 expression in these nontumorigenic S1 cells via Cx43-shRNA (Cx43KO-S1) resulted in cell cycle entry, perturbed apical polarity, mitotic spindle misorientation and loss of lumen, causing cell m ultilayering[19] and priming cells for enhanced motility and invasion[19,20] These phenotypic features observed in Cx43-KO-S1 acini represent architectural and phenotypical premalignant mammary lesions, like those observed in ductal hyperplasia in a murine m odel[23], which increase the risk of breast cancer initiation, marking Cx43-KO-S1 as a pretumorigenic culture model. The potential biomarker roles of three validated significantly dysregulated circRNAs were investigated We focused on their possible sponging capacity to their target miRNAs in the breast cancer risk progression 3D culture model and looked for matched dysregulation in the patient validation cohort. Cx43/hsa_circ_0077755/miR-182 axis, specific to Cx43 loss, was validated (Fig. 1), might serve as a biomarker signature axis for heightened-risk of breast cancer initiation and its dysregulation patterns seem to predict prognosis along breast cancer initiation and progression
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