■促炎细胞因子与1型和2型糖尿病的胰腺β细胞衰竭有关,并且已知刺激选择性RNA剪接和无义介导的RNA衰变(NMD)成分的表达。这里,我们研究了细胞因子是否调节NMD活性,并鉴定了β细胞靶向的转录同工型.
■在大鼠INS1(832/13)中瞬时表达的基于荧光素酶的NMD报告基因,人来源的EndoC-βH3或分散的人胰岛细胞用于检查促炎细胞因子(Cyt)对NMD活性的影响。NMD两个关键组成部分的功能成败,UPF3B和UPF2用于揭示细胞因子对细胞活力和功能的影响。使用标准技术部署RNA测序和siRNA介导的沉默。
■Cyt减弱产生胰岛素的细胞系和原代人β细胞中的NMD活性。发现这些效应涉及内质网应激并且与UPF3B的下调有关。通过UPF3B过表达(OE)或UPF2沉默实现的NMD活性的增加或减少提高或降低Cyt诱导的细胞死亡,分别,在EndoCβH3细胞中,与胰岛素含量降低或增加有关,分别。未观察到这些操作对葡萄糖刺激的胰岛素分泌的影响。转录组学分析显示,Cyt增加了转录同种型中的选择性剪接(AS)诱导的外显子跳跃,这可以通过UPF2沉默来增强。基因富集分析鉴定了由UPF2沉默调节的转录本,其蛋白质在细胞外基质(ECM)中定位和/或起作用。包括丝氨酸蛋白酶抑制剂SERPINA1/α-1-抗胰蛋白酶,其沉默使β-细胞对细胞毒性敏感。细胞因子通过UPR信号抑制NMD活性,可能充当针对Cyt诱导的NMD组分表达的保护性应答。
■我们的研究结果强调了RNA转换在β细胞对炎症应激反应中的重要作用。
UNASSIGNED: Proinflammatory cytokines are implicated in pancreatic ß cell failure in type 1 and type 2 diabetes and are known to stimulate alternative RNA splicing and the expression of nonsense-mediated RNA decay (NMD) components. Here, we investigate whether cytokines regulate NMD activity and identify transcript isoforms targeted in ß cells.
UNASSIGNED: A luciferase-based NMD reporter transiently expressed in rat INS1(832/13), human-derived EndoC-ßH3, or dispersed human islet cells is used to examine the effect of proinflammatory cytokines (Cyt) on NMD activity. The gain- or loss-of-function of two key NMD components, UPF3B and UPF2, is used to reveal the effect of cytokines on cell viability and function. RNA-sequencing and siRNA-mediated silencing are deployed using standard techniques.
UNASSIGNED: Cyt attenuate NMD activity in insulin-producing cell lines and primary human ß cells. These effects are found to involve ER stress and are associated with the downregulation of UPF3B. Increases or decreases in NMD activity achieved by UPF3B overexpression (OE) or UPF2 silencing raise or lower Cyt-induced cell death, respectively, in EndoC-ßH3 cells and are associated with decreased or increased insulin content, respectively. No effects of these manipulations are observed on glucose-stimulated insulin secretion. Transcriptomic analysis reveals that Cyt increases alternative splicing (AS)-induced exon skipping in the transcript isoforms, and this is potentiated by UPF2 silencing. Gene enrichment analysis identifies transcripts regulated by UPF2 silencing whose proteins are localized and/or functional in the extracellular matrix (ECM), including the serine protease inhibitor SERPINA1/α-1-antitrypsin, whose silencing sensitizes ß-cells to Cyt cytotoxicity. Cytokines suppress NMD activity via UPR signaling, potentially serving as a protective response against Cyt-induced NMD component expression.
UNASSIGNED: Our findings highlight the central importance of RNA turnover in ß cell responses to inflammatory stress.