Role of p53/circRNA0085439/Ku70 axis in DNA damage response in lung cells exposed to ZnO nanoparticles: Involvement of epigenetic regulation

Background: Nano-Zinc oxide (Nano-ZnO) has been increasingly applied in agriculture, industry and biomedicine. However, the genotoxic effects of Nano-ZnO and the underlying mechanisms remain incompletely clear. Methods: Human bronchial epithelial cell line (HBE) was used to observe the effects of Nano-ZnO on DNA damage repair-related proteins and epithelial mesenchymal transition (EMT) by Western blotting. Then, CRISPR/cas9-based technique was used to create p53 knockout (p53-KO) cell line. RNA-seq analysis was performed to uncover the circular RNA (circRNA) profile after Nano-ZnO treatment in p53-KO cells compared with p53 wild-type (p53-wt) cells. LC–MS/MS was used to discover the potential binding proteins of circRNA_0085439 in the p53 deficiency background after Nano-ZnO treatment. Nano-ZnO-induced DNA damage and EMT were also investigated in vivo by instillation of Nano-ZnO (50 µg/mouse). Results: Nano-ZnO exposure caused DNA damage and EMT at both in vitro and in vivo background, which was reflected by increased DNA damage associated proteins such as ATM and ATR and γ H2AX. p53 expression increased at the early stage post Nano-ZnO treatment decreased later. RNA-seq assay showed a highest increase of circRNA_0085439 expression in p53-KO cells compared with the p53-wt cells after Nano-ZnO exposure. Silencing of p53 expression promoted its translocation of circRNA_0085439 from cytoplasm to nucleus leading to the formation of circRNA_0085439/Ku70 complex resulting in the decreased expression of Ku70 protein. In addition, increased EMT markers, N-cadherin and Vimentin, was observed in lung epithelial cells and in mouse lungs at day 7 after Nano-ZnO exposure. Conclusions: This study unraveled the epigenetic mechanisms underlying Nano-ZnO-induced DNA damage and EMT. The effect of Nano-ZnO-induced DNA damage through p53/circRNA_0085439/Ku70 pathway likely contribute to Nano-ZnO-induced cell cytotoxicity and apoptosis. Our findings will provide information to further elucidate the molecular mechanisms of Nano-ZnO-induced cytotoxicity and genotoxicity.