Objective: Aging can lead to pancreatic islets cellular dysfunction and impaired glucose tolerance. It was unclear whether m6A mRNA methylation was involved in regulating the physiological function of elderly islet cells and its possible mechanism. Our study compared the differences in glucose metabolism, m6A regulatory key proteins and age-related proteins between elderly mice (12 months old) and young mice (3 months old), then explored effect of overexpressed YTHDF3 on pancreatic islets aging.
Methods: MeRIP-seq and mRNA-seq association analysis were used to identify the RNA methylase and the corresponding aging-related genes that were significantly different between mice of different months (12-month old mice Vs 3-month old mice). Vitro cellular aging model were established using H2O2 treatment in pancreatic islet cell line MIN-6, INS-1, and EndoC-β H1. Pancreatic islet tissues of different aged donors were acquired; then the differential expressed genes were verified by qRT-PCR and Western blotting on islet cell lines, and clinical donor islets. YTHDF3 was over expressed in MIN-6 cells, and its impact on cellular aging and possible mechanisms.
Results: Compared to 3-month old mice, 12-month old mice exhibit hyperglycemia, decreased glucose tolerance. Their islets were marked by senescence markers. Dot blotting showed an increased m6A levels in 12-month old mice pancreatic islet. MeRIP-seq and RNA-seq analysis showed different mRNA levels in Ythdf3, cell cycle checkpoint factors (Cdkn1aC, dkn2a, Ezh2 and Cdk4), aging related regulatory factors (Igf1r, Bambi), Notch signaling pathway factor Dtx1, transcription factor involved in pancreatic development (Pax6), insulin secretion related genes (Ins2 and Pdx1). Further Western blot experiments showed that YTHDF3, aging related proteins IGF1R, and BAMBI were all elevated in 12 month old mice. After treatment with H2O2, the levels of m6A in pancreatic islet cell lines were higher, YTHDF3, IGF1R and BAMBI was increased. Finally, significantly increased YTHDF3, IGF1R and BAMBI were observed in pancreatic islet tissues from elderly donors (>60 years old). When the YTHDF3 gene was over expressed in MIN-6 cells, the proliferation of these cells slowed down, aging morphology such as loose inter-cellular connections and increased expression of aging related proteins IGF1R and BAMBI were observed.
Conclusion: Our results indicated that aging-related proteins IGF1R and BAMBI were found to be modified by m6A. Aging leads to an increase in the expression of YTHDF3 in pancreatic islets. As an m6A reading protein, YTHDF3 reads m6A modifications on aging related genes, sunch as IGF1R and BAMBI, promoting the translation and expression of these genes, leading to pancreatic cell aging and downgrade its function.