Investigating the mechanisms of elevated RNA oxidation impacting pancreatic beta-cells utilizing whole transcriptome sequencing
Objective To investigate the impact of elevated glucose-induced RNA oxidation on pancreatic β-cell function,activity,and underlying molecular mechanisms.Methods Rat pancreatic islet β-cell tumour INS-1 cells were cultured in vitro and subjected to nucleic acid oxidation assessment using isotope dilution ultra-high performance liquid tandem mass spectrometry(ID LC MS/MS)following high glucose exposure.In vitro simulation of increased RNA oxidation in INS-1 cells was achieved using 8-oxoguanosine-5'-triphosphate(8-oxoGTP).Cell proliferation was evaluated through CCK-8 assay,apoptosis was measured via flow cytometry,and gene expression of insulin(INS),pancreatic-duodenal homologous cassette 1(PDX1),cysteine-aspartate proteinase 3(Casp3),and cysteine aspartate protease 6(Casp6)was analyzed at the mRNA level.Additionally,whole transcriptome sequencing was performed to elucidate the molecular mechanisms underlying the impact of RNA oxidation on INS-1 cells.Results Elevated glucose levels induced an increase in RNA oxidation within INS-1 cells.This heightened RNA oxidation led to the inhibition of INS-1 cell proliferation,a reduction in mRNA levels of INS and PDX1 genes,and the promotion of apoptosis-related casp3 and casp6 gene mRNA synthesis.Transcriptome sequencing analysis unveiled that the elevated RNA oxidation caused differential expression of mRNA,lncRNA,miRNA,and circRNA in INS-1 cells.This included a significant down-regulation of transcription factors such as Mafa,Pdx1,Pax6,and Mnx1,alongside an up-regulation of various miRNAs like rno-miR-124-3p,rno-miR-133a-3p,rno-miR-3120,rno-miR-212-3p,and rno-miR-7a-2-3p.These molecular changes contributed to the altered expression of associated lncRNAs,ultimately hindering insulin synthesis and secretion,as well as β-cell proliferation.Conclusions Increased RNA oxidation down-regulates the levels of key β-cell transcription factor mRNAs,contributes to the differential expression of related non-coding RNAs(ncRNAs),particularly lncRNAs,impacts β-cell insulin synthesis and secretion,hinders cell proliferation,and serves as a significant factor in β-cell dysfunction and decreased activity in type 2 diabetes mellitus(T2DM).
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