Circulating Exosomal MicroRNA Profiles Associated with Acute Soft Tissue Injury

Abstract

Objective: This study aimed to characterize the circulating exosomal microRNA (miRNA) profiles associated with acute soft tissue injury.

Materials and methods: In this experimental study, a total of 12 rats were randomly divided into control group and model group (n=6 for each group). The rats in the model group were used to establish an acute soft tissue injury following the mechanical injury of the leg. The exosomes from the peripheral blood of all the rats were isolated and then characterized by Nanosight NS300 particle size analyser (NTA), transmission electron microscopy (TEM) and western blot. Next, the exosomal miRNAs in the control and model groups were sequenced, and the differentially expressed miRNAs (DE-miRNAs) were identified using the DESeq algorithm. Functional analyses were performed using Gene Ontology (GO) terms and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway databases. Finally, quantitative reverse-transcription polymersa chain reaction (qRT-PCR) was used to verify the expression of the DE-miRNAs.

Results: TEM, NTA and western blot results showed that the exosomes were approximately 100 nm in size and exhibited cup-shaped morphology. A total of 628 miRNAs were obtained by sequencing. After that, 28 DE miRNAs (DEmiRNAs) were identified, including seven down-regulated miRNAs and 21 up-regulated miRNAs. These DE-miRNAs were linked to 7539 target genes with GO. Also, KEGG analyses demonstrated that these genes were enriched for phosphorylation, VEGF signaling pathway, and MAPK signaling pathway. Additionally, the consistency rate between the qRT-PCR and sequencing results was 83.33%, which showed a high relative reliability of the sequencing results.

Conclusion: These findings suggest that these 28 exosomal miRNAs may be involved in the regulation of acute soft tissue injury, by one of critical biological processes (BP), phosphorylation. The findings provide valuable clues by utilizing exosomes as therapeutic targets for the effective treatment of acute soft tissue injury.

Keywords: Exosomes; Gene Ontology; MicroRNAs; Sequencing; Soft Tissue Injuries.

Fig.1

The symptoms of acute soft tissue injury in the rat model. A. Symptoms at a micro level. B. The morphology of muscle tissues was determined by Hematoxylin-Eosin (HE) staining.

Fig.2

Characterization of exosomes from rat peripheral blood samples. A. Exosomes morphology was observed by transmission electron microscopy (TEM) (scale bar: 100 nm). B. Particle size distribution of exosomes was measured by Nanosight. C. Using western blot, exosomes surface markers (CD63, CD9, CD81) was detected.

Fig.3

Summary of small RNA sequencing data in exosomes and screening of differentially expressed RNAs (DERs). A. Pearson correlation between all samples. B. The RPKM density distribution of all samples. C. The RPKM distribution of all samples. D. The DERs volcano diagram. The blue points represent down-regulated transcripts; the grey points represent unchanged transcripts; the red points represent the up-regulated tanscripts. E. Bidirectional hierarchical clustering heatmap based on the expression level of the DERs.

Fig.4

Functional annotation of the potential target genes of DE-miRNAs. A. The predicted target genes identified using the Miranda and RNAhybrid algorithms. B-D. The GO analysis performed using DAVID. E. The signaling pathway analysis carried out using the KEGG database. DE; Differentially expressed, GO; Gene ontology, and KEGG; Kyoto encyclopedia of genes and genomes.

Fig.5

Validation of relative expression of miRNAs by qRT-PCR. A. miR-122b, B. miR-335, C. miR-206-3p, D. miR-215, E. miR-488-3p, and F. miR-342-3p. **; P<0.01 vs. the control group and qRT-PCR; Quantitative reverse-transcription polymerase chain reaction.