Deciphering the role of R-loop/RNA:DNA hybrids in tumor biology: detection, quantification and clinical relevance

R-loops/RNA:DNA hybrids (RDHs) are thermodynamically stable nucleic acid structure that are dynamically abundant in the human genome and have been linked to numerous physiological and pathological conditions. Given their regulatory significance, precise control of R-loops is crucial. However, since RDHs are difficult to detect accurately, the diagnostic and prognostic capability of RDHs have not been evaluated yet. Our study focuses on addressing this gap. We utilized cancer and non-cancer cell lines as well as tissues to optimize accurate detection of R-loop using RNase III and H enzymes. Moreover, we employed immunohistochemistry (IHC) to detect RDHs in malignant and non-malignant tissues, we showed that RDHs is associated with tumor grades, stages in different cancer types. The number of RDH enriched/specific sites (RDH-S) and RDH-S density in aggressive/advanced breast cancer tissues were significantly higher than those in low-aggressive tumors. In contrast to luminal A molecular subtype, luminal B, basal, and HER2-positive tumor cells often exhibit a larger number of RDH-S nuclei and a moderate to high nucleus RDH-S density. Quantification of the RDH-S density data revealed significant associations with clinical stages, histological grades, cell proliferation status, and lymphocyte infiltration varying across tumor types and subtypes. Furthermore, we observed a rightward shift in the nuclear RDH-S density distribution in aggressive or/and advanced cancer variants compared to the cytoplasmic RDH density distribution, highlighting RDH heterogeneity and its association with aggressiveness of cancer. Importantly, we demonstrate that the number of RDH-S positive cells, RDH-S density distribution and their sensitivity towards RNase treatment can further stratify the heterogenous histological grade 2 of breast cancer into more homogenous subgroups. Thus, the RDH/R-loop mapping and their distribution patterns in cell and tissue samples represent a prospective biomarker capable of improving cancer diagnosis, prognosis, and treatment.

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Deciphering the role of R-loop/RNA:DNA hybrids in tumor biology: detection, quantification and clinical relevance

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