Global effects of the guanine quadruplex binding ligands Pyridostatin and TMPyP4 on microRNA regulation in response to ionizing radiation in HeLa cells

Guanine quadruplexes (G4s) are secondary structures in DNA and RNA that regulate a wide range of biological processes. Small molecules that assist in formation, stabilization or destabilization of G4s indirectly alter the cellular functions governed by these structures. Pyridostatin (PDS) and TMPyP4 are two well-characterized G4-binding ligands that have been shown to modulate various cellular functions, particularly in cancer cells. However, their effects on microRNAs (miRNAs), which are key regulators of post-transcriptional gene expression, remain poorly understood. HeLa cells treated either with PDS or TMPyP4 and gamma radiation were subjected to small RNA sequencing. Analysis results revealed a higher overall small RNA content in PDS treated cells, whereas TMPyP4 treated cells exhibited a greater number of differentially expressed miRNAs following gamma radiation exposure. Target gene analysis of the top differentially regulated miRNAs revealed that molecular functions related to DNA/nucleic acid binding activities of various factors, including RNA polymerase II cis-regulatory region sequence-specific binding activity, were largely affected. Furthermore, KEGG pathway enrichment studies suggested that observed changes may result from alterations in signal transduction pathways in cells treated with G4 ligands and gamma radiation compared to untreated control cells. Additionally, G4 ligand treatment altered the cellular expression levels of histone modifying enzymes, including histone deacetylases and methyltransferases. This finding suggest that the two G4 binding ligands- PDS and TMPyP4 differentially regulate miRNA expression profiles, histone modifying enzymes and associated biological pathways in HeLa cells.