Cytoplasmic capping enzyme targeted, hypoxia-responsive RNAs, RORA and KCTD16 modulate the aggressiveness of CoCl2-induced hypoxic osteosarcoma cells

Abstract

Hypoxia is a defining feature of the solid tumour microenvironment and a major determinant of therapeutic response. Hypoxia-inducible factors (HIFs) are central regulators of transcriptional reprogramming under hypoxic stress. Hypoxia can paradoxically elicit both tumour-promoting and tumour-suppressive outcomes, suggesting regulatory mechanisms beyond canonical HIF-dependent pathways. Emerging evidence indicates that hypoxia-responsive RNAs (HRRs) may also be regulated independently of HIFs, with posttranscriptional stabilization playing a critical determinant of hypoxic adaptation. Cytoplasmic mRNA recapping mediated by the cytoplasmic capping enzyme (cCE) has recently emerged as an important post-transcriptional regulatory process, yet its role in hypoxia-driven RNA regulation remains poorly understood. Here, we aimed to identify novel HRRs that modulate cellular adaptability to hypoxia and to determine whether these transcripts are regulated by cCE. Using CoCl2-induced hypoxia, we observed a significant reduction in osteosarcoma cell aggressiveness, characterized by decreased proliferation, clonogenic survival, and migratory capacity. Transcriptomic profiling of hypoxic osteosarcoma cells identified RORA and KCTD16 as significantly upregulated and function as suppressors of tumour cell aggressiveness. Integrative in-silico CAGE tag analysis followed by cap-specific biochemical assays confirmed that both transcripts are post-transcriptionally stabilized by cCE. Mechanistically, hypoxia-induced stabilization of HIF1 transcriptionally elevated RORA and KCTD16 expression, while cCE further reinforced their stability post-transcriptionally. Stabilization of these cCE-targeted HRRs resulted in suppression of the oncogenic proliferation driver c-Myc, thereby attenuating the aggressive phenotype of hypoxic osteosarcoma cells. Collectively, our findings identify cCE as a previously unrecognized post-transcriptional regulator in hypoxia biology and reveal a RNA-centric mechanism by which hypoxia can restrain tumour aggressiveness.