Transcriptomic analysis (RNA-seq) of rat pheochromocytoma cells (PC12) treated with 20 hypoxia-reoxygenation cycles

Pheochromocytomas and paragangliomas (PPGLs) with continuous activation of hypoxia pathways in presence of oxygen (pseudohypoxia) are associated with higher disease aggressiveness. The commonly used in vitro models reflect the characteristics of these pseudohypoxic tumors only to a limited extent. We therefore hypothesized that recurrent cycles of hypoxia (hypoxia-reoxygenation cycles) lead to continuous activation of hypoxia signaling pathways under normoxic conditions and thus better reflect the pseudohypoxic phenotype of the more aggressive PPGLs. Rat pheochromocytoma (PC12) cells were treated with 20 hypoxia-reoxygenation cycles (24 h hypoxia (O2<=1%) followed by three to four days under normoxic conditions); the resulting sub-cell line was named PC12 Z20 (file names labeled with "sample"). As a control, PC12 cells were synchronously cultured under normoxic conditions (PC12 Z20 control cells; file names labeled with "sample_control"). After establishment, these new sub-cell lines (PC12 Z20 and PC12 Z20 control) were cultured for 24 h under normoxic ("sample_normoxia" or "sample_control_normoxia") or hypoxic ("sample_hypoxia" or "sample_control_hypoxia") conditions and RNA was isolated (NucleoSpin RNA Plus, Machery-Nagel GmbH, Dueren, Germany) for RNA-seq. Three different passages (R3,R4, R5) were analyzed.