Purpose: Ionizing radiation (IR) can induce autophagy and premature senescence; however, the link between them has not been clarified. Our research has shown that X-ray irradiation induces premature senescence in lung adenocarcinoma cells, and its occurrence partially depends on the signal transducer and activator of transcription 3 (STAT3). STAT3 can bind to the promoter region of Beclin1 and regulate its expression. Therefore, it is speculated that there may be a close link between premature senescence and autophagy induced by ionizing radiation in lung adenocarcinoma cells. p62 plays a regulatory role in both autophagy and premature senescence, and it is also an irreplaceable molecule that causes the senescence -associated secretory phenotype (SASP) and a substrate for selective autophagy. This study focused on STAT3, Beclin1 and p62 to clarify the regulatory relationship between IR-induced autophagy and premature senescence.
Materials and methods: After exposure to 4 Gy X-rays, a β-galactosidase staining kit was used to detect the positive rate of premature senescence. STAT3 was overexpressed by pcDNA3.0-STAT3 transfection, and was inhibited by AG490 and rapamycin. Lung adenocarcinoma cells were transduced with the adenovirus vector GV119-Beclin1 to knockdown the expression of Beclin1, or treated with ATM and ATR inhibitors to inhibit premature senescence. Western blotting was used to examine alterations in the radiation response proteins STAT3 and p-STAT3, senescence-related proteins p62 and GATA4, autophagy-related proteins Beclin1, and LC3-II/LC3-I. The mRNA expression levels of SASP factors, including IL-6 and IL-8, were examined by real-time polymerase chain reaction.
Results: The activity of SA-β-gal increased significantly (p < .05), and the expression of p62 decreased significantly at 72 h after 4 Gy X-ray irradiation, accompanied by the increased expression of STAT3, p-STAT3, Beclin1, and the LC3-II/LC3-I ratio. Up- or down-regulation of STAT3 expression was followed by an increase or decrease in Beclin1 expression. After treatment with ATM and ATR inhibitors, there were no significant changes in Beclin1 expression or LC3-II/LC3-I ratio in A549 cells after 4 Gy X-ray irradiation. The p62 expression, the percentage of the SA-β-gal-positive staining cells, and the expression of IL-6 and IL-8 mRNA in cells transduced with GV119-Beclin1 were also decreased significantly after 4 Gy X-ray irradiation compared with that of the 0 Gy group.
Conclusion: Radiation induces premature senescence and autophagy in lung adenocarcinoma cells. Autophagy regulates X-ray radiation-induced premature senescence through the STAT3-Beclin1-p62 pathway in lung adenocarcinoma cells.
Keywords: Beclin1; Radiation; STAT3; autophagy; p62-GATA4; premature senescence.