Differential Expression and Bioinformatics Analysis of Plasma-Derived Exosomal circRNA in Type 1 Diabetes Mellitus

Abstract

Backgrounds: Both exosome and circular RNA (circRNA) have been reported to participate in the pathogenesis of type 1 diabetes mellitus (T1DM). However, the exact role of exosomal circRNA in T1DM is largely unknown. Here, we identified the exosomal circRNA expression profiles in the plasma of T1DM patients and explored their potential function using bioinformatics analysis. Material and Methods. Exosomes were extracted by the size exclusion chromatography method from plasma of 10 T1DM patients and 10 age- and sex- matched control subjects. Illumina Novaseq6000 platform was used to detect the exosomal circRNA expression profiles. Multiple bioinformatics analysis was applied to investigate the potential biological functions of exosomal circRNAs.

Results: A total of 784 differentially expressed exosomal circRNAs have been identified in T1DM patients, of which 528 were upregulated and 256 were downregulated. Gene Ontology analysis enriched terms such as protein ubiquitination involved in ubiquitin-dependent protein catabolic protein (GO:0042787), membrane (GO:0016020), and GTPase activator activity (GO:0005096). The most enriched pathway in Kyoto Encyclopedia of Genes and Genomes was ubiquitin-mediated proteolysis (ko04120). The miRNA-targeting prediction method was used to identify the miRNAs that bind to circRNAs, and circRNA-miRNA-mRNA pathways were constructed, indicating that interactions between circRNA, miRNA, and gene might be involved in the disease progression.

Conclusions: The present study identified the exosomal circRNA expression profiles in T1DM for the first time. Our results threw novel insights into the molecular mechanisms of T1DM.

Figure 1

Characterization of exosomes. Transmission electron microscopy images of exosomes isolated from plasma (a). The size distribution of exosomes indicated by nanoparticle tracking analysis (b). Western blot analysis of Alix, Tsg101, CD63, and Calnexin in plasma-derived exosomes (c).

Figure 2

The overview of the exosomal circRNA sequencing data. Pie chart of circRNA type (a). Read distribution of circRNAs on different chromosomes (b). The Circos diagram of the expression level of each sample (c). Length distribution of cirRNAs (d).

Figure 3

Differential exosomal circRNA profiles in T1DM patients and healthy controls. Heatmap of the expression levels of differentially expressed exosomal circRNAs (a). Volcano plot of the identified circRNAs (b). The MA diagram of the overall distribution of exosomal circRNAs (c).

Figure 4

Biological analysis of parental genes of differentially expressed circRNAs, Top 20 Gene Ontology terms for biological processes (a), cellular component (b), and molecular function (c). Top 20 terms of Kyoto Encyclopedia of Genes and Genomes pathway analysis (d).

Figure 5

Bioinformatics analysis of identified miRNAs targeted at differentially expressed circRNAs. Top 20 Gene Ontology terms for biological processes (a), cellular component (b), and molecular function (c). Top 20 terms of Kyoto Encyclopedia of Genes and Genomes pathway analysis (d).

Figure 6

Construction of the circRNA-miRNA-mRNA competing endogenous RNA regulatory network. Diamond indicates diabetes-related gene, tip triangle indicates diabetes-related miRNA, circle indicates diabetes-related circRNA, red color indicated upregulation, and green color indicates downregulation. 8 : 37870420|37877551 (hsa_circ0005630); 20 : 47262288|47276795 (hsa_circ0007026).