Hypoxia increases the biogenesis of IGF2BP3-bound circular RNAs

Insulin-like Growth Factor 2 Binding Protein 3 (IGF2BP3) promotes cancer migration and invasion by binding to several coding and non-coding RNAs. Hypoxia stimulates tumor progression by upregulating Hypoxia Inducible Factors and downstream signaling. Quaking (QKI) gene, which is upregulated in hypoxia and promotes epithelial to mesenchymal transition (EMT), induces circular RNAs. Therefore, the axis between IGF2BP3, QKI, circular RNAs and their respective host genes under hypoxia was studied.

Several IGF2BP3-bound circular RNAs were previously identified in HepG2. There were 13 circRNAs originating from 8 host genes bound to IGF2BP3. We confirmed their binding to IGF2BP3 in U87MG using an RNA Immunoprecipitation assay. MALAT1, an oncogenic lncRNA was also found to be associated with IGF2BP3. Three adherent cell lines expressing high levels of IGF2BP3 viz., HeLa, HepG2 and U87MG were cultured under normoxia (20%O₂) and hypoxia (<0.2%O₂) for 48-168 h. Expression of IGF2BP3, QKI, EMT markers, IGF2BP3-bound circRNAs and their host mRNAs expression were assessed by quantitative real-time PCR (qRT-PCR) in both normoxia and hypoxia. The hypoxia markers viz., VEGF and CA9 were upregulated in all the cell lines in hypoxia at all time points along with an increase in SNAIL. We found 6 genes, viz., PHC3, CDYL, ANKRD17, ARID1A, NEIL3 and FNDC3B with increased expression both at the mRNA and circRNA level indicating their synergistic role in tumor initiation. Overall, we found that circRNA to mRNA expression was observed to be increased for most of the genes and time points of hypoxia in all the cell lines. IGF2BP3 and QKI were also upregulated in hypoxia indicating their role in circRNA biogenesis and stability.

Our data implies that hypoxia augments circRNA biogenesis which might subsequently play a role in tumor progression.