文献类型：期刊文献

中文题名：基于全转录组测序分析核糖核酸氧化影响胰岛β细胞的分子机制

英文题名：Investigating the mechanisms of elevated RNA oxidation impacting pancreatic beta-cells utilizing whole transcriptome sequencing

作者：周发强[1];王安奇[2];张文泽[1];文刘颖[3];张译之[3];梁玉娟[3];蔡剑平[4];王晚霞[1]

第一作者：周发强

机构：[1]兰州大学第一临床医学院,兰州730000;[2]国家卫健委胃肠肿瘤诊治重点实验室,兰州730000;[3]甘肃中医药大学第一临床医学院,兰州730000;[4]国家卫健委北京老年医学研究所,北京100730

第一机构：兰州大学第一临床医学院,兰州730000

年份：2024

卷号：43

期号：7

起止页码：889

中文期刊名：中华老年医学杂志

外文期刊名：Chinese Journal of Geriatrics

收录：CSTPCD;;北大核心:【北大核心2023】；CSCD:【CSCD2023_2024】；

基金：国家自然科学基金(81760385,82170856);国家重点研究发展计划(973计划)(2018YFC2000301);甘肃省自然基金(17JR5RA036)。

语种：中文

中文关键词：核糖核酸;胰岛β细胞;糖尿病;测序

外文关键词：RNA;Pancreatic beta cell;Diabetes;Sequencing

摘要：目的探索高糖诱导核糖核酸氧化增加对胰岛β细胞功能和活性的影响及分子机制。方法体外培养大鼠胰岛β细胞瘤INS-1细胞,高糖干预后同位素稀释超高效液相串联质谱(ID LC MS/MS)评估核酸氧化。利用8-氧代鸟苷-5'-三磷酸酯(8-oxoGTP)体外模拟INS-1细胞RNA氧化增加,CCK-8评估细胞增殖,流式细胞术评估细胞凋亡,实时荧光定量聚合酶链式反应(qRT-PCR)评估胰岛素(INS)、胰-十二指肠同源盒1(PDX1)、半胱氨酸天冬氨酸蛋白酶3(Casp3)和半胱氨酸天冬氨酸蛋白酶6(Casp6)基因信使RNA(mRNA)表达,全转录组测序分析RNA氧化影响INS-1细胞的分子机制。结果高糖致INS-1细胞RNA氧化增加。RNA氧化增加抑制INS-1细胞增殖,降低INS和PDX1基因mRNA水平,促进凋亡相关casp3和casp6基因mRNA合成。全转录组测序分析发现,RNA氧化增加导致INS-1细胞内mRNA、长链非编码RNA(lncRNA)、微RNA(miRNA)和环状RNA(circRNA)差异表达,显著下调Mafa、Pdx1、Pax6和Mnx1转录因子,上调rno-miR-124-3p,rno-miR-133a-3p,rno-miR-3120,rno-miR-212-3p,rno-miR-7a-2-3p,促使相关lncRNAs差异表达,从而抑制胰岛素合成和分泌及β细胞增殖。结论RNA氧化增加下调β细胞关键转录因子mRNAs水平,促使相关非编码RNA(ncRNA)特别是lncRNAs差异表达,影响β细胞胰岛素合成和分泌,抑制细胞增殖,是2型糖尿病(T2DM)β细胞功能障碍和活性降低的重要原因。
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,impactsβ-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).