Transcriptome profile of rat genes in injured spinal cord at different stages by RNA-sequencing

Abstract

Background: Spinal cord injury (SCI) results in fatal damage and currently has no effective treatment. The pathological mechanisms of SCI remain unclear. In this study, genome-wide transcriptional profiling of spinal cord samples from injured rats at different time points after SCI was performed by RNA-Sequencing (RNA-Seq). The transcriptomes were systematically characterized to identify the critical genes and pathways that are involved in SCI pathology.

Results: RNA-Seq results were obtained from total RNA harvested from the spinal cords of sham control rats and rats in the acute, subacute, and chronic phases of SCI (1 day, 6 days and 28 days after injury, respectively; n = 3 in every group). Compared with the sham-control group, the number of differentially expressed genes was 1797 in the acute phase (1223 upregulated and 574 downregulated), 6590 in the subacute phase (3460 upregulated and 3130 downregulated), and 3499 in the chronic phase (1866 upregulated and 1633 downregulated), with an adjusted P-value <0.05 by DESeq. Gene ontology (GO) enrichment analysis showed that differentially expressed genes were most enriched in immune response, MHC protein complex, antigen processing and presentation, translation-related genes, structural constituent of ribosome, ion gated channel activity, small GTPase mediated signal transduction and cytokine and/or chemokine activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the most enriched pathways included ribosome, antigen processing and presentation, retrograde endocannabinoid signaling, axon guidance, dopaminergic synapses, glutamatergic synapses, GABAergic synapses, TNF, HIF-1, Toll-like receptor, NF-kappa B, NOD-like receptor, cAMP, calcium, oxytocin, Rap1, B cell receptor and chemokine signaling pathway.

Conclusions: This study has not only characterized changes in global gene expression through various stages of SCI progression in rats, but has also systematically identified the critical genes and signaling pathways in SCI pathology. These results will expand our understanding of the complex molecular mechanisms involved in SCI and provide a foundation for future studies of spinal cord tissue damage and repair. The sequence data from this study have been deposited into Sequence Read Archive ( http://www.ncbi.nlm.nih.gov/sra ; accession number PRJNA318311).

Keywords: GO enrichment; Pathway analysis; RNA-Seq; Spinal cord injury; Sprague-Dawley rats (RRID:RGD_70508).

Fig. 1

BBB scores at different stages of the SCI. The BBB scores were shown at different stages of the SCI: before injury (n = 12); sham (n = 3); 1 dpi (n = 9); 3 dpi and 6 dpi (n = 6); 10 dpi, 14 dpi, 21 dpi, and 28 dpi (n = 3)

Fig. 2

PCA analysis of the expressed transcripts. PCA analysis with three principal components (PC1, 2, and 3) was performed to demonstrate the source of variance in our data (n = 3)

Fig. 3

Volcano map of differentially expressed genes. Significantly upregulated and downregulated genes are shown as a red and green dot, respectively. The blue dot represents no significant difference between the expressions of genes. a 1 dpi vs sham, b 6 dpi vs sham, c 28 dpi vs sham, d 6 dpi vs 1 dpi, e 28 dpi vs 1 dpi, f 28 dpi vs 6 dpi

Fig. 4

Quantitative RT-PCR validations of DEGs characterized by RNA-Seq. The relative expression level of target mRNAs was calculated using the ^ΔΔCt method and expressed relative to the value in the sham group (designated as 1). Data represent the mean ± SD (n = 3). Log2 fold change was the ratio of average Log2 folds between samples

Fig. 5

Hierarchical cluster analysis of gene expression in the spinal cords at different injured stages. Based on similarity of gene expression patterns, 7632 DEGs were classified into many expression cluster groups. The blue to red gradation represented gene expressions from down to up. a: sham control; b: 1 dpi; c: 6 dpi; d: 28 dpi

Fig. 6

K-means clustering of DEGs. The 7632 DEGs were statistically grouped into 8 subclusters. The trends of distinct significant expression subclusters were analyzed. A: sham control; B: 1 dpi; C: 6 dpi; D: 28 dpi. (n = 3 in every group)