Profiling of the dynamically alteredgene expression in peripheral nerve injury using NGS RNA sequencing technique

Abstract

Functional recovery of peripheral nerve injuries is of major demand in clinical practice worldwide. Although, to some extent, peripheral nervous system can spontaneously regenerate, post-injury recovery is often associated with poor functional outcome. The molecular mechanism controlling the peripheral nerve repair process is still majorly unclear. In this study, by utilizing the Next Generation Sequencing (NGS) RNA sequencing technique, we aim to profile the gene expression spectrum of the peripheral nerve repair. In total, we detected 2847 were differentially expressed at day 7 post crush nerve injury. The GO, Panther, IPA and GSEA analysis was performed to decipher the biological processes involving the differentially expressed genes. Collectively, our results highlighted the inflammatory response and related signaling pathway (NFkB and TNFa signaling) play key role in peripheral nerve repair regulation. Furthermore, Network analysis illustrated that the IL10, IL18, IFN-γ and PDCD1 were four key regulators with multiple participations in peripheral nerve repair and potentially exert influence to the repair process. The expression changes of IL10, IL18, IFN-γ, PDCD1 and TNFSF14 (LIGHT) were further validated by western blot analysis. Hopefully, the present study may provide useful platform to further reveal the molecular mechanism of peripheral nerve repair and discover promising treatment target to enhance peripheral nerve regeneration.

Keywords: Peripheral nerve repair; RNA sequencing; inflammation and NFkB.

Figure 1

Heat map analysis showing general alteration between nerve injury (L) and control group (R). Our heat map analysis revealed 2847 significant changed genes between L and R group. The two-dimensional hierarchical clustering, using the 2847 genes, clearly showed the degree of separation between L and R group.

Figure 2

Gene Ontology Enrichment Analysis (GO). We performed GO analysis which address that up regulated genes were associated with 23 classes of biological processes, while down regulated genes were associated with 27 other classes of biological processes.

Figure 3

Panther Pathway Analysis. We use Panther Pathway analysis to evaluated peripheral nerve repair regulation on cell signaling level. Up regulated genes were related with 17 pathways, while down regulated genes were only related with 4 pathways.

Figure 4

Ingenuity pathway analysis (IPA). Our IPA Data showed that the identified genes were primarily involved in NFkB signaling pathways and inflammatory response. A. Majority target genes of NFkB pathway were up regulated. B. 80 in total of 112 TNFa target genes were increased in peripheral nerve repair process. C. The overall inflammatory response is largely increased.

Figure 5

Gene Set Enrichment Analysis (GSEA). GSEA data identified subsets of 191 and 192 genes specific for inflammatory response and TNFa signaling via NFkB, respectively. The Enrichment score was 0.78 and 0.69 with p-value at <0.001 and 0.022 respectively.

Figure 6

Gene network analysis. We targeted the key “cross-road” regulator in peripheral nerve repair process using gene network analysis. Network analysis illustrated that the IL10, IL18, IFN-γ and PDCD1 were four key regulators with multiple participations in peripheral nerve repair and potentially exert influence to the repair process.

Figure 7

Westernblot verification on crossroad regulator genes. We targeted these four differentially expressed genes together with TNFSF14 (LIGHT) to verify the RNA-seq data using westernblot technique.