Nickel nanoparticle-induced cell transformation: involvement of DNA damage and DNA repair defect through HIF-1α/miR-210/Rad52 pathway
Authors: Mo Yiqun , Zhang Yue , Zhang Yuanbao , Yuan Jiali , Mo Luke , Zhang Qunwei 
Published: November 2021

Abstract
Background
Nickel nanoparticles (Nano-Ni) are increasingly used in industry and biomedicine with the development of nanotechnology. However, the genotoxic and carcinogenic effects of Nano-Ni and the underlying mechanisms are still unclear.

Methods
At first, dose–response (0, 10, 20, and 30 μg/mL) and time-response (0, 3, 6, 12, and 24 h) studies were performed in immortalized normal human bronchial epithelial cells BEAS-2B to observe the effects of Nano-Ni on DNA damage response (DDR)-associated proteins and the HIF-1α/miR-210/Rad52 pathway by real-time PCR or Western blot. Then, a Hsp90 inhibitor (1 µM of 17-AAG, an indirect HIF-1α inhibitor), HIF-1α knock-out (KO) cells, and a miR-210 inhibitor (20 nM) were used to determine whether Nano-Ni-induced Rad52 down-regulation was through HIF-1α nuclear accumulation and miR-210 up-regulation. In the long-term experiments, cells were treated with 0.25 and 0.5 µg/mL of Nano-Ni for 21 cycles (~ 150 days), and the level of anchorage-independent growth was determined by plating the cells in soft agar. Transduction of lentiviral particles containing human Rad52 ORF into BEAS-2B cells was used to observe the role of Rad52 in Nano-Ni-induced cell transformation. Nano-Ni-induced DNA damage and dysregulation of HIF-1α/miR-210/Rad52 pathway were also investigated in vivo by intratracheal instillation of 50 µg per mouse of Nano-Ni. gpt delta transgenic mice were used to analyze mutant frequency and mutation spectrum in mouse lungs after Nano-Ni exposure.

Results
Nano-Ni exposure caused DNA damage at both in vitro and in vivo settings, which was reflected by increased phosphorylation of DDR-associated proteins such as ATM at Ser1981, p53 at Ser15, and H2AX. Nano-Ni exposure also induced HIF-1α nuclear accumulation, miR-210 up-regulation, and down-regulation of homologous recombination repair (HRR) gene Rad52. Inhibition of or knocking-out HIF-1α or miR-210 ameliorated Nano-Ni-induced Rad52 down-regulation. Long-term low-dose Nano-Ni exposure led to cell malignant transformation, and augmentation of Rad52 expression significantly reduced Nano-Ni-induced cell transformation. In addition, increased immunostaining of cell proliferation markers, Ki-67 and PCNA, was observed in bronchiolar epithelial cells and hyperplastic pneumocytes in mouse lungs at day 7 and day 42 after Nano-Ni exposure. Finally, using gpt delta transgenic mice revealed that Nano-Ni exposure did not cause increased gpt mutant frequency and certain DNA mutations, such as base substitution and small base insertions/deletions, are not the main types of Nano-Ni-induced DNA damage.

Conclusions
This study unraveled the mechanisms underlying Nano-Ni-induced cell malignant transformation; the combined effects of Nano-Ni-induced DNA damage and DNA repair defects through HIF-1α/miR-210/Rad52 pathway likely contribute to Nano-Ni-induced genomic instability and ultimately cell transformation. Our findings will provide information to further elucidate the molecular mechanisms of Nano-Ni-induced genotoxicity and carcinogenicity.

Graphical Abstract