Abstract:

Objective

To analyze the mechanism of secoisolariciresinol diglucoside （SDG） affecting apoptosis and ferroptosis of non-small cell lung cancer A549 cells through circular RNA homeodomain interacting protein kinase 3 （circRNA HIPK3）.

Methods

Lung cancer A549 cells were divided into A549 group，SDG-L group，SDG-M group，SDG-H group，SDG-H+oe-NC group，SDG-H+oe-circRNA HIPK3 group.Each experiment was repeated 6 times. The 3-（4，5-dimethylthiazol-2-yl）-2，5-diphenyltetrazolium bromide assay was used to detect the cell viability. The mRNA expressions of circRNA HIPK3，nicotinamide adenine dinucleotide phosphate oxidase 1 （NOX1），glutathione peroxidase 4 （GPX4），ferritin heavy chain 1 （FTH1），and solute carrier family 7 member 11 （SLC7A11）. Kits were used to detect the levels of Fe²⁺，malondialdehyde （MDA），and glutathione （GSH）. The 2'，7'-dichlorofluorescin diacetate staining method was used to detect the level of reactive oxygen species （ROS）. Western blot was used to determine the expression changes of NOX1，GPX4，FTH1，SLC7A11，nuclear factor erythroid 2-related factor 2 （Nrf2），and heme oxygenase-1 （HO-1）. The Transwell assay and terminal deoxynucleotidyl transferase dUTP nick end labeling assay were used to detect cell invasion and apoptosis.

Results

SDG at different concentrations had no significant effect on the survival rate of human bronchial epithelial cells HBE1，but it decreased the survival rate of A549 cells. The cell viability in the SDG-H group ［（55.29±10.05） %］ decreased most significantly，and 40 μmol/L of SDG was selected for subsequent experiments. Circular RNA HIPK3 was highly expressed in the cells of the A549 group （2.36±0.33） and lowly expressed in the cells of the SDG-H group （1.72±0.26）.The levels of Fe²⁺，MDA，ROS and the cell apoptosis rate in the SDG-H group were higher than those in the A549 group （P＜0.01）. The mRNA and protein expressions of NOX1 were higher than those in the A549 group（P＜0.01）. The levels of GSH and the number of cell invasions （P＜0.05）. The mRNA and protein expressions of GPX4，FTH1，SLC7A11，and the protein levels of Nrf2 and HO-1 were lower than those in the A549 group（P＜0.05）. The levels of Fe²⁺，MDA，ROS and the cell apoptosis rate in the cells of the SDG-H+oe-circRNA HIPK3 group were lower than those in the SDG-H+oe-NC group （P＜0.05）. The mRNA and protein expressions of NOX1 were lower than those in the SDG-H+oe-NC group （P＜0.05）. The levels of GSH and the number of cell invasions were higher than those in the SDG-H+oe-NC group（P＜0.05）. The mRNA and protein expressions of GPX4，FTH1，SLC7A11，and the protein levels of Nrf2 and HO-1 were higher than those in the SDG-H+oe-NC group （P ＜0.05）.

Conclusion

SDG promoted apoptosis of non-small cell lung cancer A549 cells and inhibited cell invasion by regulating circRNA HIPK3. This might be related to inhibiting the activation of the Nrf2/HO-1 antioxidant pathway and activating ferroptosis in cells.

Key words: Bronchogenic carcinoma, Secoisolariciresinol diglucoside, Circular RNA homeodomain-interacting protein kinase 3, Apoptosis, Ferroptosis