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

BackgroundNano-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.MethodsHuman 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 mu g/mouse).ResultsNano-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 gamma 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.ConclusionsThis 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.

Automated summary

This study revealed the epigenetic mechanisms underlying Nano-ZnO-induced DNA damage and EMT. The effect of Nano-ZnO-induced DNA damage through the p53/circRNA_0085439/Ku70 pathway likely contributes to Nano-ZnO-induced cell cytotoxicity and apoptosis. These findings provide important information to further elucidate the molecular mechanisms of Nano-ZnO-induced cytotoxicity and genotoxicity.