Comprehensive IsomiR sequencing profile of human pancreatic islets and EndoC-βH1 beta-cells

AbstractAims/HypothesisMiRNAs play a crucial role in regulating the islet transcriptome, influencing beta cell functions and pathways. Emerging evidence suggests that during biogenesis a single miRNA locus can generate various sequences, known as isomiRNAs (isomiRs). However, a comprehensive profiling analysis of isomiRs in human pancreatic islets and beta cells is still lacking. This study aims to unveil the isomiRs expression profile in Laser Capture Microdissected (LCM) human pancreatic islets (HI) from non-diabetic donors and in the human beta cell line EndoC-βH1, in order to shed light on novel molecular mechanisms governing beta cell function.MethodsRNA was extracted from LCM HI deriving from n=19 non-diabetic donors and from EndoC-βH1 beta cells. Small RNA-seq was performed. Data were processed with the sRNAbench online pipeline for miRNAs/isomiRs quantification. Results were further validated using an external miRNA-seq database (isomiRdb).ResultsIn both HI and EndoC-βH1, isomiRs accounted for a substantial proportion of total miRNA reads (HI: 59.4+/-1.9%; EndoC-βH1: 43.8+/-0.6%). Among isomiRs, the most prevalent types were 3’-end modifications, including trimming (HI=71.8+/-2.8%; EndoC-βH1=55.8+/-1.0%) and extension (HI: 12.1+/-1.9%; EndoC-βH1: 17.4+/-0.9%), followed by non-templated addition (HI: 9.8+/-0.9%; EndoC-βH1: 14.0+/-1.2%). The analysis of the composition of the n=10 most expressed miRNAs highlighted a significant contribution of reads assigned to isomiRs. For instance, the most abundant miRNA, miR-375-3p, resulted from 59.7+/-2.4% of canonical and 40.3+/-2.4% of isomiRs in EndoC-βH1 and from 45.3+/-2.0% of canonical and 54.7+/-2.0% of isomiR reads in HI. Interestingly, miR-7-5p, a beta cell-specific miRNA, was predominantly expressed as an isomiR both in EndoC-βH1 (65.3+/-2.7%) and in HI (82.4+/-1.4%). To identify a reliable beta cell isomiR signature, common sequences detected in HI and EndoC-βH1 were filtered based on their contribution to total miRNA expression, ultimately resulting in a set of 46 isomiRs. The expression of the isomiR signature in beta cells was further evaluated using an external database, isomiRdb, which contains small-RNA sequencing data from 99 different human cell types. This analysis revealed a significant enrichment of 11 out of the 46 isomiRs in beta cells compared to other cell types. The signature was functionally characterized through regression analysis with clinical and metabolic parameters related to beta cell function in non-diabetic individuals, demonstrating a significant negative correlation between basal insulin secretion and isomiR-411-5p, but not with its corresponding canonical miRNA.Conclusion/InterpretationThis study provides a comprehensive profile of isomiR expression in pancreatic islets and beta cells, highlighting the potential significance of isomiRs as novel regulators of beta cell function.Graphical AbstractResearch in ContextWhat is already know about this subject?- isomiRs are sequence variants of microRNAs (miRNAs) and may have distinct functional role respect to the canonical sequence.- isomiRs show cell/tissue specificity and are involved in multiple disease contexts.What is the key question?- What is the profile of isomiRs in human pancreatic islets (HI) and in beta cells?- Do isomiRs have a functional role in beta cells?What are the new findings?- IsomiRs represent a relevant fraction of total miRNAs in HI and beta cells.- 3’end miRNA sequence modifications are the major fraction of isomiRs in beta cells.- A set of n=11 isomiRs, more expressed than their canonical miRNAs, are enriched in beta cells compared to the other human cell types.- Specific isomiRs are associated with beta cell glucose sensitivity and basal insulin secretion.How might this impact on clinical practice in the foreseeable future?- A comprehensive profile of isomiRs in beta cells may improve our understanding of molecular mechanisms driving beta cell function and dysfunction.- A highly detailed and granular view of miRNAs sequence variants and their expression levels may help in the design of novel therapeutic RNA-based strategies.