The Role Of M~6A Methylation In The Regulation Of CHAC1 In Inorganic Arsenic-induced Ferroptosis Of Pancreatic β Cells

Objective:Inorganic arsenic is one of the common environmental pollutants.A large number of studies have shown that exposure to inorganic arsenic is closely related to various metabolic diseases,including type 2 diabetes(T2D).Pancreaticβcells are essential for human insulin secretion,and their pathological changes play an important regulatory role in the occurrence and development of T2D.Our previous studies have shown that exposure to inorganic arsenic can lead to ferroptosis of pancreaticβcells,which in turn affects the function of pancreaticβcells.However,the mechanism by which inorganic arsenic inducesβ-cell ferroptosis remains unclear.N6-methyladenosine(m~6A)modification is the most common and essential modification in RNA post-transcriptional modification.It regulates the splicing,nuclear export,translation and degradation of RNA and plays a vital role in the occurrence and development of diseases.However,the role of m~6A methylation in inorganic arsenic-inducedβ-cell ferroptosis remains unclear.Therefore,this study will explore the mechanism of inorganic arsenic-induced ferroptosis inβcells and the regulatory role of m~6A methylation in it.Methods:In this study,C57BL/6J mice were used as the in vivo experimental model,and min6 cells were used as the in vitro experimental model.In the in vivo experiments,C57BL/6J mice were randomly divided into control and exposure groups.The control group was given a regular diet and drinking water,and the exposed group was given inorganic arsenic exposure(0 and 4 mg/L)by drinking water.After 12 weeks of treatment,pancreatic tissues were collected.The expression levels of the ferroptosis-related proteins glutathione peroxidase 4(GPX4)and acyl-Co A synthetase long-chainfamilymember4(ACSL4),glutathione-specific gamma-glutamylcyclotransferase 1(CHAC1)and glutamate-cysteine ligase catalytic subunit(GCLC)and m~6A methylation-related enzymes in pancreatic tissue were detected by Western blot.In the in vitro experiment,min6 cells were exposed to inorganic arsenic at a dose of 4μM for 24 h.The expression levels of ferroptosis-related proteins GPX4 and ACSL4 were detected by Western blot;the lipid reactive oxygen species(lipid ROS)level was detected by immunofluorescence.The expression levels of CHAC1 and GCLC were detected by Western blot,and the gene expression level of CHAC1 was detected by reverse transcription PCR(q RT-PCR).Small interfering RNA(si RNA)technology was used to interfere with the expression of CHAC1 in min6 cells,and Western blot was used to detect the expression of ferroptosis-related proteins in min6 cells;immunofluorescence was used to detect the level of lipid ROS in min6 cells.The expression of methyltransferase METTL3 in min6 cells was intervened by si RNA,and the protein expression level of CHAC1 in min6 cells was detected by Western blot.Results:The results of in vivo experiments showed that the expression levels of ferroptosis-related protein GPX4 in mouse pancreatic tissues were significantly decreased after arsenic exposure,while the expression levels of ACSL4 were increased considerably.We also found that the glutathione catabolic enzyme CHAC1 expression levels significantly increased in pancreatic tissues after inorganic arsenic exposure,while the expression levels of synthetase GCLC were not significantly altered.In addition,inorganic arsenic exposure could substantially promote the expression level of m~6A methyltransferase METTL3.In vitro experiments showed that inorganic arsenic exposure could significantly boost the occurrence of ferroptosis in min6 cells.The expression level of GPX4 was significantly decreased,and ACSL4 was significantly increased.Meanwhile,inorganic arsenic exposure could promote the production of lipid ROS in min6 cells.In addition,consistent with the results of in vivo experiments,inorganic arsenic exposure significantly promoted the protein expression level of glutathione catabolic enzyme CHAC1,while the expression level of synthetase GCLC was also not significantly altered.q RT-PCR results indicated that inorganic arsenic exposure significantly promoted the gene expression level of CHAC1.After the intervention of CHAC1 expression,we found that the downregulation of GPX4expression level and the upregulation of COX-2 expression level caused by inorganic arsenic exposure were significantly alleviated,while the level of lipid ROS in min6cells was significantly reduced,indicating that inorganic arsenic-inducedβ-cell ferroptosis was inhibited.Finally,our intervention on METTL3 expression revealed that inhibition of METTL3 expression could effectively suppress the inorganic arsenic-induced upregulation of the CHAC1 protein expression level.It indicates that inorganic arsenic may regulate m~6A methylation through METTL3,which promotes the protein expression level of CHAC1.Conclusion:Inorganic arsenic may promote the expression of CHAC1 by regulating m~6A methylation,thereby enhancing the decomposition of glutathione,affecting the expression level of GPX4,and finally leading to the occurrence of ferroptosis in pancreatic β cells.