Circulating exosomal lncRNA contributes to the pathogenesis of spinal cord injury in rats

Abstract

Exosome-derived long non-coding RNAs (lncRNAs) are extensively engaged in recovery and repair of the injured spinal cord, through different mechanisms. However, to date no study has systematically evaluated the differentially expressed lncRNAs involved in the development of spinal cord injury. Thus, the aim of this study was to identify key circulating exosome-derived lncRNAs in a rat model of spinal cord injury and investigate their potential actions. To this end, we established a rat model of spinal cord hemisection. Circulating exosomes were extracted from blood samples from spinal cord injury and control (sham) rats and further identified through Western blotting and electron microscopy. RNA was isolated from the exosomes and sequenced. The enrichment analysis demonstrated that there were distinctively different lncRNA and mRNA expression patterns between the two groups. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Gene Ontology (GO) functional analysis were performed to determine the possible involvements of upregulated and downregulated lncRNAs in various pathways and different biological processes, as well as their cellular locations and molecular functions. Furthermore, quantitative reverse transcription-polymerase chain reaction showed that the expression of five lncRNAs--ENSRN0T00000067908, XR_590093, XR_591455, XR_360081, and XR_346933--was increased, whereas the expression of XR_351404, XR_591426, XR_353833, XR_590076, and XR_590719 was decreased. Of note, these 10 lncRNAs were at the center of the lncRNA-miRNA-mRNA coexpression network, which also included 198 mRNAs and 41 miRNAs. Taken together, our findings show that several circulating exosomal lncRNAs are differentially expressed after spinal cord injury, suggesting that they may be involved in spinal cord injury pathology and pathogenesis. These lncRNAs could potentially serve as targets for the clinical diagnosis and treatment of spinal cord injury.

Keywords: exosome; inflammation; lncRNA; mRNA; miRNA; microenvironment; spinal cord injury; spinal cord repair.

Figure 1

Experimental design. CG: Control group; EG: experimental group; GO: Gene Ontology; HE: hematoxylin and eosin; KEGG: Kyoto Encyclopedia of Genes and Genomes; lncRNA: long non-coding RNA; qRT-PCR: quantitative reverse transcription-polymerase chain reaction.

Figure 2

Establishment of a rat model of spinal cord injury and evaluation by functional score and hematoxylin and eosin staining. (A) Modified Tarlov’s scale scores. n = 3 for each group. ***P < 0.001, vs. CG. Data are shown as means ± standard deviation (SD) from more than three statistical independent experiments. (B) Hematoxylin and eosin staining of normal spinal cord and injured spinal cord sites. Scale bars: 200 nm. CG: Control group; EG: experimental group.

Figure 3

Extraction and identification of circulating exosomes. (A) Representative images of Western blotting of the isolated exosomes for exosome markers. (B) Staining enhanced the clarity of the exosome membrane structure, as seen by electron microscopy. An exosome with typical structure is indicated by the white arrows. Scale bars: 200 nm. CG: Control group; EG: experimental group; HSP70: heat shock protein 70.

Figure 4

lncRNA and mRNA expression is significantly different after spinal cord injury. (A) Heat map of differentially expressed lncRNAs. (B) Two-dimensional presentation of a cluster analysis of differentially expressed lncRNAs. (C) Heat map of differentially expressed mRNAs. (D) Two-dimensional presentation of a cluster analysis of differentially expressed mRNAs. Red represents relatively high expression, green represents relatively low expression, and black represents average expression. P < 0.05, fold change > 2.0. CG: Control group; EG: experimental group; lncRNA: long non-coding RNA.

Figure 5

KEGG pathway analysis and GO functional analysis of upregulated circulating lncRNAs after spinal cord injury. (A) KEGG pathway analysis of upregulated lncRNAs. (B–D) GO functional analysis of upregulated lncRNAs. Biological process, cellular component, and molecular function analyses of upregulated lncRNAs are shown in B, C, and D, respectively. GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; lncRNA: long non-coding RNA.

Figure 6

KEGG pathway analysis and GO functional analysis of downregulated circulating lncRNAs after spinal cord injury. (A) KEGG pathway analysis of downregulated lncRNAs. (B–D) GO functional analysis of downregulated lncRNAs. Biological process, cellular component, and molecular function analyses of downregulated lncRNAs are shown in B, C, and D, respectively. GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; lncRNA: long non-coding RNA.

Figure 7

Verification of differentially expressed lncRNAs by quantitative reverse transcription-polymerase chain reaction. (A) Relative expression of upregulated lncRNAs, including ENSRN0T00000067908, XR_590093, XR_591455, XR_360081, and XR_346933. (B) Relative expression of downregulated lncRNAs, including XR_351404, XR_591426, XR_353833, XR_590076, and XR_590719. *P < 0.05, **P < 0.01, ***P < 0.001. Data are shown as means ± standard deviation (SD) from more than three statistically independent experiments. CG: Control group; EG: experimental group; lncRNA: long non-coding RNA.

Figure 8

lncRNA-miRNA-mRNA coexpression network. This interaction network is centered around 10 differentially expressed lncRNAs, which are represented by green polygons. These lncRNAs were coexpressed with and interacted with 41 miRNAs, which are indicated by red triangles, and 198 mRNAs, which are indicated by blue boxes. lncRNA: Long non-coding RNA.