Transcriptome analysis of molecular mechanisms underlying facial nerve injury repair in rats

Abstract

Although the transcriptional alterations inside the facial nucleus after facial nerve injury have been well studied, the gene expression changes in the facial nerve trunk after injury are still unknown. In this study, we established an adult rat model of facial nerve crush injury by compressing the right lateral extracranial nerve trunk. Transcriptome sequencing, differential gene expression analysis, and cluster analysis of the injured facial nerve trunk were performed, and 39 intersecting genes with significant variance in expression were identified. Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway analyses of the 39 intersecting genes revealed that these genes are mostly involved in leukocyte cell-cell adhesion and phagocytosis and have essential roles in regulating nerve repair. Quantitative real-time polymerase chain reaction assays were used to validate the expression of pivotal genes. Finally, nine pivotal genes that contribute to facial nerve recovery were identified, including Arhgap30, Akr1b8, C5ar1, Csf2ra, Dock2, Hcls1, Inpp5d, Sla, and Spi1. Primary Schwann cells were isolated from the sciatic nerve of neonatal rats. After knocking down Akr1b8 in Schwann cells with an Akr1b8-specific small interfering RNA plasmid, expression levels of monocyte chemoattractant protein-1 and interleukin-6 were decreased, while cell proliferation and migration were not obviously altered. These findings suggest that Akr1b8 likely regulates the interaction between Schwann cells and macrophages through regulation of cytokine expression to promote facial nerve regeneration. This study is the first to reveal a transcriptome change in the facial nerve trunk after facial nerve injury, thereby revealing the potential mechanism underlying repair of facial nerve injury. This study was approved by the Animal Ethics Committee of Nantong University, China in 2018 (approval No. S20180923-007).

Keywords: Akr1b8; Gene-Act Networks; RNA-Seq; Schwann cells; cell proliferation; facial nerve injury; inflammatory response; transcriptomics analysis.

Figure 1

Establishment of a facial nerve crush injury model. (A) Diagram of the transcriptome study of facial nerve injury. (B–E) Neurological scores at different times after facial nerve crush injury. All data are shown as the mean ± SD (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001, vs. control group (0 hours) (Student’s t-test). Accurate P-values for Figure 1B–E are listed in Additional Table 8. GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; ns: non-significant; STC: series test of cluster.

Figure 2

DEG analysis in the facial nerve trunk following crush injury. (A) The detailed expression pattern of 299 genes in profile 9. All of the 299 genes were up-regulated at 6 hours post-injury and down-regulated from 4 to 7 days post-injury. (B) Biological process analysis of the 299 genes. (C) DEGs at different time points post-injury. The Venn diagram shows that a total of 246 genes were differentially expressed at all time points examined post-injury. (D) The intersection between DEGs and genes in profile 48. The Venn diagram shows that 39 genes were common to both the 246 DEGs and the 299 genes of profile 48. DEGs: Differentially expressed genes.

Figure 3

Functional enrichment of the 39 specific differentially expressed genes in Figure 2D. (A) Heat map of expression levels of the 39 specific genes during the time course studied. The color scale shown on the top represents the relative expression level of the indicated genes: red indicates expression > 0, and blue indicates expression < 0. (B) GO enrichment analysis of the 39 specific genes. (C) Plot of KEGG enrichment of the significant pathways involving the 39 specific genes. The horizontal axis represents the enrichment score. The ordinate represents the functional description of the enriched pathway. A larger circle indicates that more genes were enriched for this function. The color spectrum ranging from blue to red represents the uncorrected P-value. (D) Co-expression network of the 39 specific genes. Cycle nodes in the network indicate genes, and edges between two nodes indicate interactions between genes. GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes.

Figure 4

Verification of expression of the key genes. (A) Quantitative reverse transcription-polymerase chain reaction estimation of mRNA expression of the indicated genes at different time points post-injury. All data are shown as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, vs. control group (0 hours) (Student’s t-test). (B) Schematic diagram showing transcriptional changes at 14 days after facial nerve crush injury. The schematic diagram describes the nine key genes and their potential mechanisms of regulating facial nerve regeneration. ns: Non-significant.

Figure 5

The effect of Akr1b8 knockdown on SC phenotype. (A) The mRNA expression level of Akr1b8 in SCs transfected with siNC or Akr1b8-specific siRNA (siAkr1b8-1, siAkr1b8-2, siAkr1b8-3) (n = 3) (B, C) Effect of Akr1b8 knockdown on cell proliferation in SCs. (B) The cells were fluorescently stained with EdU (red), and the nucleus was stained with Hoechst 33342 (blue). SCs transfected with siAkr1b8-2 displayed less EdU-positive (red) signal than SCs transfected with siNC. Scale bar: 100 µm. (C) Quantitation of the EdU-positive cells (n = 3). (D, E) Effect of Akr1b8 knockdown on cell migration in SCs. Wound healing assays are shown in D; both the siAkr1b8-2 group and siNC group displayed no cell migration in the clear area at 0 hours, while the siAkr1b8-2 group showed a slower migration rate than the siNC group at 12 hours and displayed a larger clear area. The quantitation data are shown in E (n = 3). Scale bar: 100 µm. (F, G) Effect of Akr1b8 knockdown on the synthesis of induced chemokines in SCs. The mRNA expression levels of MCP-1 (F) and IL-6 (G) in SCs transfected with siNC or Akr1b8-specific siRNA (siAkr1b8-2, siAkr1b8-3) are shown (n = 3). All data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, vs. siNC group (Student’s t-test). IL-6: Interleukin-6; MCP-1: monocyte chemoattractant protein-1; ns: non-significant; SC: Schwann cell; EdU: 5-ethynyl-20-deoxyuridine, siNC: negative-control siRNA.