IsomiRs in Beta Cells: Unveiling Post-Transcriptional Modifications in Functional and Inflamed States

IsomiRs are post-transcriptional modifications of canonical microRNA (miRNA) sequences. These modifications are often overlooked in research, which typically considers miRNAs as canonical sequences rather than as a diverse pool of variants. However, the biogenesis of these modifications is linked to cellular stress and inflammation, providing valuable insights into the underlying mechanisms of these processes, particularly in the context of inflammatory diseases like Type 1 diabetes (T1D). In this study, we first profiled isomiRs in four beta cell sources: (I) laser capture microdissected human islets (LCM-HI), (II) collagenase-isolated human islets (HI), (III) sorted beta cells, and (IV) EndoC-BetaH1 beta cell line to investigate their role in beta cell function. Using a state-of-the-art bioinformatics pipeline for isomiR analysis, we demonstrated the conservation and specificity of these post-transcriptional modifications in beta cells compared to other cell types. Furthermore, by integrating metabolic profiling from the LCM-HI cohort with isomiRs expression, we observed several significant associations, in particular for isomiR-411-5p-Ext5'. This specific isomiR was negatively correlated with basal and total insulin secretion rate, unlike its canonical counterpart. Additional studies on isomiR-411-5p-Ext5' revealed that it reduces glucose-stimulated insulin secretion in the EndoC-BetaH1 and specifically downregulates genes involved in vesicle budding and trafficking. These findings suggest that isomiR-411-5p-Ext5' plays a role in insulin processing and trafficking. Next, we analyzed isomiRs expression in beta cells under inflammatory stress to mimic Type 1 Diabetes (T1D) conditions. Data from EndoC-BetaH3 beta cell line and human pancreatic islets, both untreated and treated with a cytokine mix (Interferon-gamma, Interleukin-1 beta, and/or Tumor Necrosis Factor-alpha), were analyzed to explore miRNAs post-transcriptional modification in the context of T1D. We developed a novel analysis based on the proportions of different isomiR classes within miRNAs to identify potential alterations in isomiR composition at the single-miRNA level. This approach revealed a consistent mechanism of miRNAs 3' end trimming upon inflammation across the datasets. RNA sequencing analysis on HI identified a subset of exoribonucleases potentially involved in this mechanism, with the Interferon Stimulated Gene (ISG20) validated as upregulated in EndoC-BetaH3 cells under inflammatory stress. ISG20 expression was also significantly associated with the observed miRNA 3' end trimming phenomenon. Collectively, these findings highlight the importance of isomiRs in beta cells, where they play a specific role in regulating cell function under physiological conditions. Additionally, they undergo distinct post-transcriptional modifications upon inflammation, offering valuable insights into the processes regulating both beta cell function and dysfunction.