m6A methylation pattern of SLC7A11 mediates the effects of cinobufagin on hepatocellular carcinoma cell proliferation and radioresistance

To determine whether and how cinobufagin regulates hepatocellular carcinoma (HCC) cell proliferation and radioresistance.

Radiosensitive (HepG2-NC) and radioresistant (HepG2-SR) HCC cells were treated with cinobufagin, X-ray ionizing radiation (IR) or a combination of cinobufagin and IR at different doses. Cell counting was performed using the Cell Counting Kit-8 assay. Key ferroptosis marker levels were determined using the indicated methods. RNA immunoprecipitation using an anti-m6A antibody followed by quantitative polymerase chain reaction was performed to determine the m6A levels in SLC7A11 mRNA.

Cinobufagin inhibited the proliferation of HepG2-NC and HepG2-SR cells. Exposure to X-rays decreased the HepG2-NC cell count in a time- and dose-dependent manner, but did not affect HepG2-SR cells. A low dose of cinobufagin did not change the cell count without IR exposure, but re-sensitized HepG2-SR cells to IR. The combination of low-dose cinobufagin and IR increased ferroptosis and decreased SLC7A11 expression levels. Mechanistically, the combination of a low dose of cinobufagin and IR decreased m6A levels in the 3' UTR of SLC7A11 in a METTL3-dependent manner.

A low dose of cinobufagin exerted synergistic effects with IR and re-sensitized radioresistant HCC cells to IR via a METTL3/m6A-dependent pathway.