R. Zhu1, S. Cai2, and Y. Tian1; 1Department of Radiotherapy and Oncology, The Second Affiliated Hospital of Soochow University, Suzhou, China, 2Department of Radiotherapy and Oncology, The Second Affiliated Hospital of Soochow University, SuZhou, China
Purpose/Objective(s): There is currently no effective strategy available to mitigate radiation-induced intestinal injury (RIII). Because of its central role in the DNA damage response, p53 is often referred to as the ‘guardian of the genome’. When activated in an oscillatory manner, it promotes repair of DNA damage, but sustained activation can lead to cell death. The aim of this study was to investigate the regulatory mechanism of p53 oscillatory activation in intestinal stem cells (ISCs) following radiation exposure. Additionally, a mathematical model of the protein regulatory network was used to predict the most effective method of drug delivery to promote p53 oscillatory activation. Materials/
Methods: Male C57BL/6J mice were exposed to 14Gy of whole abdominal irradiation (WAI). Immunofluorescence (IF) staining was used to observe the expression of p53 protein in crypts at various time points after WAI. To assess the mRNA and m6A levels of genes associated with the p53 pathway, we utilized RNA-seq, MeRIP-seq, and MeRIP-PCR. Additionally, we identified the murine double minute 2 (MDM2) N6-methyladenosine (m6A)-modified methylase through RNA-pulldown, mass spectrometry analysis, RIP, and dual luciferase reporter gene assays. A mathematical model of the p53 regulatory network was constructed, incorporating methyltransferase-like protein 3 (mettl3), and used to predict the effects of a mettl3 inhibitor (S-Adenosylhomocysteine, SAH) on p53 activation. To assess the impact of SAH, we measured the number of germinations, surface area, and survival rate of mouse intestinal crypt organoids. We also observed the survival and body weight. Furthermore, we performed immunofluorescence tests for intestinal stem cell markers and p53. Results: After WAI, it was observed that P53 expression in mouse crypts occurred in an oscillatory pattern. Further analysis of sequencing data revealed that MDM2, the most important negative feedback regulator of p53, is regulated by m6A. This was confirmed through subsequent experiments, which demonstrated the binding of mettl3 to MDM2 mRNA. Knockdown of mettl3 resulted in a significant decrease in MDM2 mRNA, m6A, and protein levels in HIEC-6 cells after irradiation, providing evidence for the regulation of MDM2 by mettl3. Mathematical modeling of the p53 regulatory network showed that administering the appropriate dose of SAH resulted in an elevated level of p53 oscillatory activation, leading to an increase in the germination rate, surface area, and survival rate of irradiated organoids, as well as an improvement in the survival and body weight of mice. Furthermore, both organoid and mouse models showed an increase in the number of stem cells and a decrease in the amount of DNA damage. Conclusion: Overall, the present study shows that IR-responsive mettl3 is involved in IR-induced DNA damage repair in ISCs, probably by regulating the p53 oscillatory activation via MDM2 m6A modification, which may be a novel mechanism involved in the occurrence and development of RIII.
