The Mechanism And Intervention Strategy Of RRNA 6-methyladenine Modification In Radiation-induced Intestinal Injury

[Background]Nuclear radiation and radiation therapy are two major risk factors for radiation-induced intestinal injury.Before Chernobyl nuclear leakage and Hiroshima atomic bomb event caused a large area of nuclear radiation,which is a major risk factor for radiation-induced intestinal injury.On the other hand,X-ray was discovered by Rontgen in 1895 and applied to clinical practice in 1896.Radiotherapy is one of the main treatments for patients with pelvic tumors.However,intestinal injury is the main radiotherapy-related complication for the clinical application and efficacy of pelvic tumors.m6A modification,in which the hydrogen（H）atom of the nitrogen（N）at position 6is replaced by a methyl group on the adenosine of mRNA,is the most characteristic mRNA modification.m6A is a dynamic reversible process mainly involved by Writer（methyltransferase）,Eraser（demethyltransferase）and Reader（reading protein）.Most of the m6A methylation on mRNA is catalyzed by the Writer complex,which is mainly composed of the core subunits METTL3 and METTL14,WTAP,VIRMA,ZC3H13,HAKAI,RBM15/15B.Eraser mainly removes m6A methylation on mRNA,mainly including FTO and ALKBH5.Reader mainly reads m6A modifications on mRNAs,which mainly include YTH family proteins.Previous studies have reported that METTL3and N6-methylated adenine promote the repair of double-stranded DNA damage mediated by homologous recombination by regulating the accumulation of DNA-RNA heterozygous junctions at double-stranded DNA damage sites.This suggests that radiation-induced intestinal damage may affect the changes in the level of m6A modification.Our previous study found that the level of m6A modification and its key enzyme METTL14 were increased in radiation-induced intestinal injury.Therefore,this suggests that m6A modification may play an important role in radiation-induced intestinal injury.At present,the widely used small molecule drugs of key enzymes of m6A modification mainly include METTL3 and FTO inhibitors.STM2457 is a highly efficient and selective inhibitor of RNA methyltransferase METTL3 with high specificity for METTL3.FB23-2 and Dac51 are potent and selective FTO inhibitors that bind to FTO and selectively inhibit its demethylase activity.More and more studies have shown that the level of m6A modification is closely related to the occurrence of diseases.Our previous study found that decreased level of m6A modification promoted radiation-induced intestinal injury.Therefore,this suggests that changing the level of m6A modification using small molecule drugs may play a role in radiation intestinal injury.[Aims]To construct a radiation-induced intestinal injury model,and to explore the changes of m6A modification and key enzyme levels in radiation-induced intestinal injury.[Methods]To construct a simulated radiation-induced intestinal injury model,C57 wild-type male mice were irradiated with X-rays at doses of 0,16,18,and 20Gy,respectively.The body weight,diarrhea,and survival of the mice were recorded.The appropriate dose was found to establish a model of radiation-induced intestinal injury.The body weight,diarrhea and survival of the mice were recorded.The intestinal injury of the mice was obtained by analyzing the number of surviving cryps by HE staining.The level of m6A modification and the changes of key enzymes were detected to confirm whether m6A modification was involved in radiation-induced intestinal injury.To establish a model of simulated nuclear radiation intestinal injury,C57 wild-type male mice were irradiated with 0 and 9Gy of ⁶⁰Co ray,respectively and the body weight,diarrhea,and survival of the mice were recorded.The intestinal injury of the mice was obtained by analyzing the number of surviving cryps by HE staining.The level of m6A modification and the levels of key enzymes were detected to confirm whether m6A modification was involved in radiation-induced intestinal injury.By constructing a model of radiation intestinal injury at the molecular level,intestinal epithelial specific METTL14 knockout mice were generated by crossing Villi Cre mice with METTL14 flox/flox mice.The body weight,diarrhea,and survival of the mice were recorded after the radiation intestinal injury model was established by using X-rays.The intestinal injury of mice was detected by intragastric administration of FITC-dextran.Finally,the role of m6A modification level in radiation-induced intestinal injury was determined by intestinal epithelium-specific knockout METTL14 mice.At the same time,the small molecule inhibitor of METTL14 was used to reduce the level of METTL14 in the mouse small intestinal organoid.The mouse small intestinal organoid model was established by X-ray irradiation,and the role of m6A modification level in radiation-induced intestinal injury was determined by the growth state of the organoid.After experimental determination of the role of m6A modification in radiation-induced intestinal injury,wild-type male C57 mice were intraperitoneally injected with FTO inhibitors Dac51 and FB23-2 for 5 consecutive days to increase the overall level of m6A modification in mice.On the 6th day,the mice were irradiated with X-rays to establish a model of radiation intestinal injury.The intestinal injury of mice was evaluated by analyzing the number of surviving crypts by HE staining.[Results]The results showed that the 0,16,and 18Gy groups had less intestinal injury and rapid self-recovery by analyzing the body weight and the number of surviving crypts.The 20Gy group had more severe intestinal injury and no death,which was suitable for subsequent experiments.After 20Gy irradiation,the body weight of the mice was significantly reduced,and the intestinal injury was severe according to the statistical analysis of the number of surviving crypts after HE staining.At the molecular level,the levels of m6A modification and its key enzyme METTL14 were increased.In the ⁶⁰Co simulated nuclear radiation model,the body weight of the mice was significantly reduced after 9Gy irradiation,and the intestinal injury was serious according to the statistical analysis of the number of surviving crytis after HE staining.At the molecular level,the level of m6A modification and its key enzyme METTL14increased.There was no significant difference in body weight between wild-type mice and gut-specific METTL14 knockout mice,and no diarrhea or death occurred in each group of mice.Intestinal permeability was significantly enhanced in gut-specific METTL14knockout mice compared with wild-type mice as detected by FITC-dextran in serum after gavage.At the same time,the small molecule inhibitor of METTL14 was used to reduce the level of METTL14 in the small intestinal organoids of mice and X-rays were used to construct a radiation injury model.Compared with the control group,the organoids in the SAH inhibitor group collapsed earlier and more severely.The model of radiation-induced intestinal injury was established by increasing the level of intestinal m6A modification with FTO inhibitor.Compared with the control group,the intestinal injury was significantly reduced after increasing the overall level of intestinal m6A modification with FTO inhibitor.[Conclusion]In radiation-induced intestinal injury,the level of m6A modification and the level of its key enzyme METTL14 are increased.After inhibiting the level of m6A modification in the intestine of mice,radiation-induced intestinal injury is aggravated.The administration of FTO inhibitor increases the level of intestinal m6A modification and effectively protects the radiation-induced intestinal injury.