Rationale: Type 1 diabetes (T1D) is characterised by autoimmune destruction of pancreatic β-cells. Enterovirus (EV) infections have been frequently linked to T1D, but a causal relationship has not been well-established. MicroRNAs (MiRNAs) function as post-transcriptional regulators of gene expression. However, their role in virus-induced β-cell death has not been investigated. We hypothesize that EV infection of β-cells alters both cellular and circulating miRNAs abundance, thereby inducing specific gene expression patterns which result in β-cell death and T1D pathogenesis. Objective: To investigate the association between EV infection and miRNA dysregulation in human islets infected with EVs, and to develop circulating miRNA signatures to detect and distinguish EV-infection in T1D patients. Methods and Results: (a) Primary human islets were infected with clinical EV strains, including Coxsackievirus (CV)A9, CVB2, CVB5, isolated from stool specimens of children at seroconversion to islet autoimmunity. Cells/cell culture supernatant were collected at D0-to-D14 post-viral infection to measure viral replication, cytopathic effect (CPE) and cellular miRNAs; (b) Plasma samples from children with T1D and/or EV infection were tested for circulating miRNAs among different between-group comparisons (EVpositive/T1D; EVnegative/control; EVpositive/control and EVnegative/T1D). RNA was extracted using (a) miRVana kit and (b) Trizol-based method. All samples were tested using global miRNA profiling (TaqMan OpenArray, QuantStudio12K Flex). In addition, 11 individual miRNA were tested (Quantitative realtime-PCR, TaqMan). 'miRBase', 'TargetScan' and 'miRWalk' miRNA databases were used to identify validated and predicted miRNA gene targets. DAVID Functional Annotation tool was used for gene-enrichment analysis of the putative target genes. All EV clinical isolates replicated and demonstrated CPE in human islets. Differential expression of individual miRNAs was demonstrated in the infected islets vs no-virus control (NVC) islets. The expression of miRNA-627, miRNA-302a, miRNA-190b, miRNA-497, miRNA-888, miRNA-124* and miRNA-340* were significantly higher in CVB2-infected islets vs NVC (> 10 fold difference). In addition, subsets of circulating miRNAs were found to be differentially expressed between the study groups. Of 434 miRNAs detected in at least one plasma sample, 11 miRNAs were significantly different between the 4 groups and between T1D vs controls (miR-539, miR-532, miR-886-5p, miR-125a-5p, miR-340, miR-574-3p, miR-28-3p, miR-150, miR-339-3p, miR-151-3p) (p-value <0.05). MiRs miR-376a, miR-629, miR-140, miR-345, miR-146b, miR-222, miR-146a were also significantly different between T1D vs Controls. miR-139-5p was significantly altered across the 4 groups, and between EV-positive vs EV-negative groups alongside miR-136*, miR-744*, miR-15a*, miR-148a, miR-379, miR-410, miR-223*, miR-93*, miR-342-3p, miR-885-5p, miR-520c-3p. Moreover, 13 miRNAs were detected in all samples and groups in the initial study, however only miR-151-3p was significantly differentially expressed. Gene-enrichment analysis revealed apoptotic and inflammatory cytokine signaling to be major biological pathways that are regulated by the miRNAs detected. Conclusions: Viral infection of human islets leads to dysregulation of multiple miRNAs that may have a critical role in immune modulation, thereby contributing to β-cell death in T1D. Such EV-associated T1D may also be detected in altered circulating miRNAs. Measuring circulating and cell-associated miRNAs will greatly further our understanding of virus-induced T1D, which could have broad applicability as a disease biomarker.


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