Comparative Proteomic and Microarrays analysis to identify molecular changes associated to APE1/Ref‐1 loss of expression.

APE1/Ref‐1 is essential for cell protection toward oxidative stress by acting as a transcriptional regulator of antioxidant genes and as a central factor in base excision repair pathways of DNA lesions. While it is known that loss of APE1/Ref‐1 expression is associated to significant biological effects, the molecular mechanisms involved in these processes are still not completely known. By using conditional gene expression knockdown by RNA interference (RNAi) technology in HeLa cell line, we inspected the molecular changes associated to APE1/Ref‐1 loss of expression by both proteomics and microarrays analysis. Loss of APE1/Ref‐1 expression was associated with significant increase in apoptosis, stop of cell growth, accumulation of abasic DNA damage, increase in intracellular ROS formation and oxidative stress‐induced cellular damage. Proteomics analysis allowed the identification of five proteins upregulated (i.e. Annexin A3, 3′(2′),5′‐bisphosphate nucleotidase 1, ATP‐dependent RNA helicase DDX39, Chloride intracellular channel protein 3) and four proteins downregulated (i.e. Rab GDP dissociation inhibitor beta, Guanine deaminase, Coatomer subunit epsilon, Microtubule associated protein RP/EB family member 1) upon APE1/Ref‐1 silencing. Microarrays data analysis showed the involvement of 1126 genes whose expression was significantly altered (550 upregulated and 576 downregulated) upon APE1/Ref‐1 silencing. The majority of these genes are associated with cell cycle and apoptosis or with cell adhesion and signal transduction. This work represents the basis for future studies on the comprehension of the causal role played by APE1/Ref‐1 in regulating different biological mechanisms through the control of gene expression.