Global Analysis Of Transcriptome In Dorsal Root Ganglia Following Peripheral Nerve Injury In Rats And The Role Of Vav1 On Neurite Outgrowth

Part I Global analysis of transcriptome in dorsal root ganglia following peripheral nerve injury in ratsObjectives:To obtain a global perspective of changes following nerve injury and elucidate the mechanisms underlying nerve regeneration.Methods:1. Establishment of rat sciatic nerve crush injury model.2. By RNA sequencing, we detected transcriptional changes in dorsal root ganglion(DRG) neurons at 0 h, 3 h, 9 h, 1 d, 4 d and 7 d following sciatic nerve crush injury in rats.3. Differentially expressed genes were then selected and classified into major clusters according to their expression patterns.4. In order to know credible biological functions, we conducted the Gene Ontology(GO) analyses and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis regarding the intersected clusters.5. Gene act networks were then constructed for the intersected clusters and the expression of pivotal genes of gene act networks was validated by quantitative real-time PCR.Results:1. In the L4-L6 DRGs, by RNA-Seq, we have identified 1688 genes differentially expressed during the different time points post nerve injury.2. Two major clusters of the significantly altered genes during the time course post nerve injury were selected for further bioinformatic analyses and we found that ion transport, cellular ion homeostasis, calcium ion transport, regulation of neuron projection development, defense response, regulation of cell death, response to bacterium and regulation of cell activation were enriched following nerve injury.3. Gene act networks based on KEGG database were constructed and most of the gene interactions were involved in Signal transduction, Nervous system, Sensory system,Immune system, Signal transduction, Signaling molecules and interaction, and Cellular community.4. Refer to the interaction degree of genes and the KEGG pathway analyses, we selected several genes in cluster 3 and cluster 6 to validate their expression during peripheral nerve injury by q RT-PCR. q RT-PCR and RNA-seq data were highly correlated to each other.Conclusions:We have performed the first RNA-seq transcriptome profiling study of rat DRGs following sciatic nerve crush injury. The results showed that at 9 h, an early stage after nerve injury, a set of genes involved in Transcript factors, Immunity, G-protein receptor,Calcium signaling pathway and Apoptosis was dysregulated. While, at 1 d, a later stage after nerve injury, signal pathways involved in Trascription, Cell differentiation and development, Cell adhesion molecules, Axoanl outgrowth and Growth factors were activated. These results provide further insights on the molecular changes following peripheral nerve injury in a transcriptional landscape, facilitating to elucidate the biological process and molecular mechanisms of peripheral nerve injury.Part II The role of Vav1 on neurite outgrowth and the interaction network of its expressionObjectives:To find the key regulate genes in DRG neuron outgrowth, reveal the role of Vav1 on neurite outgrowth and explore its mechanism.Methods:1. According to the validation of RNA-Seq data and bioinformatic analyses, we selected some genes for further functional research in DRG neurons in vitro.2. By quantitative real-time PCR, we detected the m RNA expression alteration of Vav1 following sciatic nerve crush injury in rats.3. We performed Western blotting to detect the protein expression alteration of Vav1 following sciatic nerve crush injury in rats.4. Immunofluorescence analysis of Vav1 expression and distribution in DRG sections.5. We dissociated DRG neurons in vitro and interfered Vav1 expression in DRG neurons. The efficiency of Vav1 silencing was measured by quantitative real-time PCR.And with axon specific antibody Tuj1, we detected neurite outgrowth.6. We used IPA software to analysis possible gene interaction network of Vav1 following sciatic nerve crush injury in rats.Results:1. By quantitative real-time PCR, we confirmed that the m RNA expression of Vav1 was increased following sciatic nerve crush injury in rats, consistent with previous RNA-seq result.2. Western blot analyses shown that Vav1 expression was increased following nerve injury in DRG sections.3. Immunofluorescence analysis of Vav1 expression showed that Vav1 was indeed expressed in DRG neurons and altered its expression following nerve injury.4. quantitative real-time PCR shown that si RNA-1 and si RNA-3 of Vav1 could significantly decrease Vav1 expression in DRG neurons. And silencing Vav1 expression may lead to the decline of DRG neurite outgrowth.5. IPA analysis shown that, at different time points following nerve injury, there were several genes of significant differential expression, may interact with Vav1 and constructed into an interaction network that is concerned with nerve regeneration.Conclusions:1. Vav1 is up-regualted following peripheral nerve injury, especially at 4 d;2. Immunostaining showed that Vav1 was expressed in DRG neurons and its expression was increased after nerve injury.3. In vitro si RNA silencing results showed that interfering Vav1 expression could inhibited DRG neuron outgrowth significantly;4. Following nerve injury, Vav1 could interact with some key genes, such as IL6, Jun,SH2B2 and EZH2, at different time points;In conclusion, Vav1 might be a key functional gene in nerve injury and regeneration.