YTHDF3 Is Involved In The Regulation Of Physiological Function Of Aging Islet Cells Through M6A-mRNA Methylation

Objective:China has entered an aging society,and impaired glucose tolerance and diabetes in the elderly threaten the quality of life and life expectancy of the elderly.It is increasingly urgent to elucidate the mechanism of the physiological function of islet cells in the elderly.In our previous work,we found that the expression of YTHDF3 was significantly different from that of control mice in islets transcriptome sequencing,and YTHDF3 mainly affected the translation of m6A modifier genes.By exploring the role of m6A-mRNA methylation in the regulation of physiological function of islet cells in the elderly,this study provides a basis for potential targeted therapy for m6A-binding protein,and provides new ideas for the diagnosis,prevention and treatment of diabetes in the elderly.Methods:MeRIP-seq and mRNA-seq association analysis were used to identify the RNA methylase YTHDF3 and the corresponding aging-related genes Igf1r and Bambi that were significantly different between mice of different months.The differentially expressed genes were verified by quantitative real-time PCR（q RT-PCR）and Western blotting（WB）on islet cell lines,mouse islets,and clinical donor islets.In vitro（senescing cell model and cell overexpression assay）and in vivo（mouse islets of different months and human islets of different ages）experiments were performed to confirm the involvement of YTHDF3 in the regulation of islet cell senescence.The cell cycle related proteins(p16^INK4a,p21^CIP1,EZH2,CDK4)were further verified by WB,and the cell cycle related genes（Cdkn2a,Cdkn1a,Ezh2,Cdk4）were verified by q RT-PCR.Results:At the age of 12 months,the mice developed insulin resistance and the islets were marked by senescence markers.RNA-seq analysis of isolated islets of the two groups of mice showed that the transcription level of m6A reading protein YTHDF3 was significantly increased,and differential gene enrichment analysis showed that the most significant expression difference was in ribosomal pathway related genes.The combined mRNA-seq and MeRIP-seq analysis showed that the genes changed in both omics were involved in aging-related genes including Igf1r and Bambi.In vitro and in vivo experiments confirmed that YTHDF3 was involved in the regulation of islet cell senescence.Cell cyclin-related proteins(p16^INK4a,p21^CIP1,EZH2,CDK4)were further verified by WB,and it was confirmed that the islets of aging mice were in a state of cell arrest and the physiological function of islets was affected.Conclusion:In this study,the aging-related proteins IGF1R and BAMBI were found to be modified by m6A.When the m6A reading protein YTHDF3 is increased,the senesence-related proteins are also increased,indicating that YTHDF3 promotes the expression of proteins by reading m6A modification on related genes and causes islet cells to enter the senescence state.Islet cell cycle proteins are regulated,cell cycle arrest,and physiological function of islet cells is affected.Our study provides the basis for targeted therapy against m6A regulators to protect islet cell survival and function.