C. Liu1, S. Cai2, Y. Tian3, and R. Zhu4; 1Department of Radiotherapy and Oncology, The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China, 2Department of Radiotherapy and Oncology, The Second Affiliated Hospital of Soochow University, SuZhou, China, 3Institute of Radiotherapy & Oncology, Soochow University, Suzhou, China, 4Department of Radiotherapy and Oncology, The Second Affiliated Hospital of Soochow University, Suzhou, China
Purpose/Objective(s): The high radiosensitivity of intestinal epithelium is the main limiting factor for the effect of radiotherapy for abdominal and pelvic malignant tumors. Unfortunately, there is no effective prevention or treatment strategy to mitigate radiation-induced intestinal injury (RIII). At present, studies show that direct DNA damage and oxidative stress are the two major pathogenesis of radiation-induced intestinal injury. Sestrin-2 (SESN2) is a conservative antioxidant protein. The expression of SESN2 will be up-regulated due to oxidative stress, which is a mechanism to compensate the cell damage by the way of inhibiting ROS and activating autophagy. The aim of this study is to provide a new target for the prevention and treatment of radiation-induced intestinal injury by studying the function and molecular mechanism of SESN2 in the process of radiation-induced intestinal injury repair. Materials/
Methods: Male C57BL/6J mice were exposed to 14Gy of whole abdominal irradiation (WAI) to establish a mouse model of radiation-induced intestinal injury. The levels of mRNA and m6A of genes in small intestine after irradiation were observed by RNA-seq, MeRIP-seq and MeRIP-PCR. Through a series of experiments such as the Cell Counting Kit 8, flow cytometry, ?H2AX immunofluorescence and comet assays in vivo and in vitro, we studied the radiosensitivity of intestinal epithelium irradiated with the knockout or overexpression of SESN2 and detect of DNA damage and antioxidant reduction indexes in nucleus and mitochondria. Furthermore, the molecular mechanism of the interaction between SESN2 and m6A reader IGF2BP2 was analyzed by RNA pull-down, mass spectrometry analysis, RIP and dual luciferase reporter gene assays. Results: The analysis of sequencing data revealed that SESN2 is regulated by m6A.The expression level of SESN2 in human intestinal epithelial cells is regulated by ionizing radiation. In vivo and in vitro experiments, its overexpression can promote radiation resistance. Through the detection of DNA damage and antioxidant reduction indexes, we found that SESN2 can regulate ROS production both in nucleus and mitochondria. Through RNA pull-down, mass spectrometry analysis, RIP, we found that SESN2 is regulated by m6A reader IGF2BP2 after ionizing radiation. And there is a decrease in survival rate and weight mice after inhibiting IGF2BP2 in vivo. Finally, we conducted the recovery experiment. Knockdown of SESN2 with overexpression of IGF2BP2 resulted in an increase in SESN2 mRNA, m6A, and protein levels and reducing DNA damage and production of ROS in HIEC-6 cells after irradiation, providing evidence for the regulation of SESN2 by IGF2BP2. Conclusion: In conclusion, SESN2 depends on m6A modification of IGF2BP2 and further regulates the production of ROS in the nucleus and mitochondria, which may be a novel mechanism involved in the occurrence and development of RIII.
