Transcription Profiling of a Revealed the Potential Molecular Mechanism of Governor Vessel Electroacupuncture for Spinal Cord Injury in Rats

Abstract

Objective: This study aimed to identify differentially expressed genes (DEGs) by transcriptome analysis to elucidate a potential mechanism by which governor vessel electroacupuncture (GV-EA) promotes neuronal survival, axonal regeneration, and functional recovery after complete transection spinal cord injury (SCI).

Methods: Sham, control, or GV-EA group adult female Sprague Dawley rats underwent a complete transection SCI protocol. SCI area RNA-seq investigated the DEGs of coding and noncoding RNAs 7 days post-SCI. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were used to classify DEGs functions, to explain a possible molecular mechanism. Immunofluorescence and BBB (Basso, Beattie, and Bresnahan) score were used to verify a GV-EA treatment effect following SCI.

Results: GV-EA treatment could regulate the expression of 173 mRNA, 260 lncRNA, and 153 circRNA genes among these DEGs resulted by SCI. GO enrichment analysis showed that the DEGs were most enriched in membrane, actin binding, and regulation of Toll-like receptor signaling pathway. KEGG pathway analysis showed enriched pathways (e.g. , Toll-like receptors, MAPK, Hippo signaling). According to the ceRNA network, miR-144-3p played a regulatory role by interacting with lncRNA and circRNA. GV-EA also promoted the injured spinal cord neuron survival, axonal regeneration, and functional improvement of hind limb locomotion.

Conclusion: Results of our RNA-seq suggest that post-SCI GV-EA may regulate characteristic changes in transcriptome gene expression, potential critical genes, and signaling pathways, providing clear directions for further investigation into the mechanism of GV-EA in subacute SCI treatment. Moreover, we found that GV-EA promotes neuronal survival, nerve fiber extension, and motor function recovery in subacute SCI.

Keywords: Bioinformatic analysis; CeRNA; Governor vessel electroacupuncture; RNA sequencing; Spinal cord injury.

Fig. 1.

Differential mRNA expression, gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. (A) Heatmap of differentially expressed genes (DEGs) between sham (n=3), control (n=5), and governor vessel electroacupuncture (GV-EA) (n=5) groups (p<0.05) with green and red indicating downregulated and upregulated expression, respectively. (B, C) Venn diagrams show DEGs overlap among experimental groups. Green and red represent downregulated and upregulated expression, respectively. (D) Top 30 GO terms in the enrichment analysis. The abscissa represents numbers of differentially expressed mRNAs and the ordinate represents the function in each GO term. Pink: BP; Green: cellular component; Blue: molecular function. (E) Bubble diagram of top 20 significantly enriched KEGG pathways. The abscissa shows the rich factor and ordinate shows the enrichment to KEGG pathways. The circle size marks the number of genes and the color change from red to blue indicates p-value change from low to high.

Fig. 2.

Differential lncRNA expression in the injured spinal cord of sham (n=3), control (n=5), and governor vessel electroacupuncture (GV-EA) (n=5) groups. (A) Heatmap of differentially expressed genes (DEGs) between sham, control, and GV-EA samples (p<0.05) with green and red indicating downregulated and upregulated expression, respectively. (B, C) Venn diagrams show overlaps of DEGs between experimental groups. Green and red represent downregulated and upregulated expression, respectively.

Fig. 3.

Differential circRNA expression in the injured spinal cord of sham (n=3), control (n=5), and governor vessel electroacupuncture (GV-EA) (n=5) groups. (A) Heatmap of differentially expressed genes (DEGs) between sham, control, and GV-EA samples (p<0.05) with green and red indicating downregulated and upregulated expression, respectively. (B, C) Venn diagram show overlaps of DEGs among experimental groups. Green and red represent downregulated and upregulated expression, respectively.

Fig. 4.

CeRNA network construction. (A) LncRNA-miRNA-mRNA ceRNA network. Red ball, green circle and orange triangle indicate mRNA, miRNA and lncRNA, respectively. (B) CircRNA-miRNA-mRNA ceRNA network. Red ball, green circle, and blue diamond indicate mRNA, miRNA, and circRNA, respectively.

Fig. 5.

Governor vessel electroacupuncture (GV-EA) can protect neurons survival, promote axon regeneration and improve function recovery. (A, B) The immunofluorescence staining of NeuN and NF in sham, control, and GV-EA group after spinal cord injury (SCI) 4 weeks, scale bars=1,000 µm; 40 µm in (a1–a3; b1–b3). (C) Bar chart shows the number of NeuN+ and Hoe⁺ in the area of spinal cord injury. (D) Bar chart shows the mean fluorescence intensity of NF positive expression in the area of spinal cord injury. (E) Basso, Beattie, and Bresnahan (BBB) behavioral scores in the different stages of sham, control, and GV-EA group after SCI 4 weeks. n=5/group; ^*p<0.05 indicates significant difference when compared with the sham group. ^#p<0.05 indicates significant difference when compared with the control group.