Investigation of Cx43-Derived Exonic hsa_circ_0077755 and Exon-Intronic hsa_circ_0077753/miRNA-182 Interaction: Functional Impact on Early Breast Cancer Initiation and Identification of Circulating circRNA/miRNA Signatures

Abstract

Circular RNAs (circRNAs) have emerged as essential regulators of gene expression through their ability, in part, to sponge miRNAs. Yet, the specific roles of different circRNA subtypes, especially exonic versus exon-intronic isoforms, in the earliest stages of breast cancer remain underexplored. GJA1, which encodes the gap-junction protein Connexin-43 (Cx43) crucial for epithelial communication, gives rise to 3 circRNAs, including the exonic hsa_circ_0077755 and the exon-intronic hsa_circ_0077753. In this work, we first functionally dissected these two circRNAs in non-tumorigenic S1 human breast epithelial cells by transiently and stably downregulating each. We profiled their reciprocal regulation to onco-miR-182 by RT-qPCR and dual-luciferase assays, and we assessed resulting phenotypes through 3D lumen formation, β-catenin distribution, and invasion ability. We efficiently silenced both circRNAs, but they produced non-equivalent phenotypes. Compared with hsa_circ_0077755 knockdown, even with the associated reduction in Cx43 mRNA and protein, hsa_circ_0077753 knockdown produced a more pronounced tumor-initiating phenotype, disrupted lumen formation, disorganized β-catenin, and increased invasion ability. Both circRNAs deregulated miR-182 expression, but loss of hsa_circ_0077753 produced a more pronounced increase in miR-182 and decreased hsa_circ_0008932, another circRNA predicted by bioinformatics analysis to sponge miR-182. In line with this pronounced miR-182 shift, only the hsa_circ_0077753-KD reduced the downstream miR-182 tumor suppressor targets BCL2, FOXO1, and RECK, consistent with a more robust tumor-initiation signature. Notably, extracellular vesicles mirrored the expression changes of hsa_circ_0077753, hsa_circ_0077755, hsa_circ_0008932, and miR-182 that we observed in the cells, suggesting that released vesicles carry the same circRNA-miRNA signature as their cells of origin. Building on this, we next asked whether such reciprocal circRNA/miRNA expression could enable minimally invasive early detection of breast cancer. We hypothesize that during early tumor initiation, alterations in the expression of oncogenes or tumor suppressor targets mirror similar changes in the levels of corresponding circular RNAs and reciprocal changes in the expression of the corresponding sponged miRNAs. We designed a bioinformatics pipeline to define mRNA/circRNA/miRNA axes that could be detected in blood. Using TCGA, we selected stage I breast cancer patients aged 20-50 and identified differentially expressed genes and miRNAs associated with the earliest epigenetic imprints of breast cancer. We then integrated these with differentially expressed circRNAs from GSE182471 and intersected circRNA parental genes with TCGA differentially expressed genes (DEGs) to identify circRNAs likely involved in early tumorigenesis. For each circRNA, we retrieved predicted miRNA interactors (CircInteractome) and verified their clinical relevance using Kaplan-Meier Plotter, UALCAN, and EnrichR. This yielded 19 candidate mRNA/circRNA/miRNA axes identified from breast cancer tissue datasets, with the potential to be translated into circulating, blood-detectable biomarkers for early detection of breast cancer. As part of our ongoing work, we are developing a computational algorithm designed to process large and continually expanding RNA-seq, circRNA, and miRNA sequencing data. This algorithm dynamically integrates data across platforms and self-updates with newly available datasets, enabling continuous refinement of the identified regulatory axes. Such a system evolves, increases the robustness of biomarker identification, and potentially scales to other cancer types beyond breast cancer whenever data is available. The incorporation of artificial intelligence and automated data integration enhances the algorithm's ability to extract clinically relevant insights from multi-omics data. We propose that monitoring both circRNAs and their partnered miRNAs, rather than either alone, in at-risk but still healthy individuals, could provide an earlier, blood-based window on breast tumor initiation, in line with the differential functional roles we uncovered for Cx43-derived circRNAs.