Objective

A widely recognized metabolic side effect of glucocorticoid (GC) therapy is steroid-induced diabetes mellitus (DM). However, studies on the molecular basis of GC-induced pancreatic beta cell dysfunction in human beta cells are lacking. The significance of non-coding RNAs in various cellular processes is emerging. In this study, we aimed to show the direct negative impact of GC on beta cell function and elucidate the role of riborepressor GAS5 lincRNA in the GC signaling pathway in human pancreatic beta cells.

Methods

Patients undergoing two weeks of high-dose prednisolone therapy were monitored for C-peptide levels. Human pancreatic islets and the human beta cell line EndoC-βH1 were incubated in pharmacological concentrations of dexamethasone. The GAS5 level was modulated using anti-sense LNA gapmeR or short oligonucleotides with GAS5 HREM (hormone response element motif). Immunoblotting and/or real-time PCR were used to assess changes in protein and RNA expression, respectively. Functional characterization included glucose-stimulated insulin secretion and apoptosis assays. Correlation analysis was performed on RNAseq data of human pancreatic islets.

Results

We found reduced C-peptide levels in patients undergoing high-dose GC therapy. Human islets and the human beta cell line EndoC-βH1 exposed to GC exhibited reduced insulin secretion and increased apoptosis. Concomitantly, reduced expression of important beta cell transcription factors, PDX1 and NKX6-1, as well as exocytotic protein SYT13 were observed. The expression of the glucocorticoid receptor was decreased, while that of serum and glucocorticoid-regulated kinase 1 (SGK1) was elevated. The expression of these genes was found to significantly correlate with GAS5 in human islet transcriptomics data. Increasing GAS5 levels using GAS5 HREM alleviated the inhibitory effects of dexamethasone on insulin secretion.

Conclusions

The direct adverse effect of glucocorticoid in human beta cell function is mediated via important beta cell proteins and components of the GC signaling pathway in an intricate interplay with GAS5 lincRNA, a potentially novel therapeutic target to counter GC-mediated beta cell dysfunction.

中文翻译：

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糖皮质激素通过核阻遏物 GAS5 LincRNA 诱导人类 β 细胞功能障碍。

客观的

糖皮质激素 (GC) 治疗的一个广泛认可的代谢副作用是类固醇诱发的糖尿病 (DM)。然而，关于GC诱导的人β细胞胰腺β细胞功能障碍的分子基础的研究还很缺乏。非编码 RNA 在各种细胞过程中的重要性正在显现。在本研究中，我们旨在展示GC对β细胞功能的直接负面影响，并阐明核阻遏物GAS5 lincRNA在人胰腺β细胞GC信号通路中的作用。

方法

监测接受两周大剂量泼尼松龙治疗的患者的 C 肽水平。将人胰岛和人 β 细胞系 EndoC-βH1 在药理学浓度的地塞米松中孵育。使用反义 LNAgapmeR 或带有 GAS5 HREM（激素反应元件基序）的短寡核苷酸来调节 GAS5 水平。免疫印迹和/或实时PCR分别用于评估蛋白质和RNA表达的变化。功能表征包括葡萄糖刺激的胰岛素分泌和细胞凋亡测定。对人类胰岛的 RNAseq 数据进行相关分析。

结果

我们发现接受高剂量 GC 治疗的患者 C 肽水平降低。暴露于 GC 的人胰岛和人 β 细胞系 EndoC-βH1 表现出胰岛素分泌减少和细胞凋亡增加。与此同时，重要的 β 细胞转录因子 PDX1 和 NKX6-1 以及胞吐蛋白 SYT13 的表达减少。糖皮质激素受体的表达降低，而血清和糖皮质激素调节激酶1（SGK1）的表达升高。发现这些基因的表达与人类胰岛转录组数据中的 GAS5 显着相关。使用 GAS5 HREM 增加 GAS5 水平减轻了地塞米松对胰岛素分泌的抑制作用。

结论

糖皮质激素对人 β 细胞功能的直接副作用是通过重要的 β 细胞蛋白和 GC 信号通路的成分与 GAS5 lincRNA 的复杂相互作用介导的，GAS5 lincRNA 是对抗 GC 介导的 β 细胞功能障碍的潜在新治疗靶点。