Identification of adhesion-associated DNA methylation patterns in the peripheral nervous system

Abstract

Schwann cells are unique glial cells in the peripheral nervous system. These cells provide a range of cytokines and nutritional factors to maintain axons and support axonal regeneration. However, little is known concerning adhesion-associated epigenetic changes that occur in Schwann cells after peripheral nerve injury (PNI). In the present study, adhesion-associated DNA methylation biomarkers were assessed between normal and injury peripheral nerve. Specifically, normal Schwann cells (NSCs) and activated Schwann cells (ASCs) were obtained from adult Wistar rats. After the Schwann cells were identified, proliferation and adhesion assays were used to assess differences between NSCs and ASCs. Methylated DNA immunoprecipitation-sequencing and bioinformatics analysis were used to identify and analyze the differentially methylated genes. Reverse transcription-quantitative PCR was performed to assess the expression levels of adhesion-associated genes. In the present study, the proliferation and adhesion assays demonstrated that ASCs had a more robust proliferative activity and adhesion compared with NSCs. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed to identify methylation-associated biological processes and signaling pathways. Protein-protein interaction network analysis revealed that Fyn, Efna1, Jak2, Vav3, Flt4, Epha7, Crk, Kitlg, Ctnnb1 and Ptpn11 were potential markers for Schwann cell adhesion. The expression levels of several adhesion-associated genes, such as vinculin, BCAR1 scaffold protein, collagen type XVIII α1 chain and integrin subunit β6, in ASCs were altered compared with those in NSCs. The current study analyzed adhesion-associated DNA methylation patterns of Schwann cells and identified candidate genes that may potentially regulate Schwann cell adhesion in Wistar rats before and after PNI.

Keywords: DNA methylation; Gene Ontology; Kyoto Encyclopedia of Genes and Genomes; Schwann cells; bioinformatics; cell adhesion; nerve regeneration; peripheral nerve injuries; protein-protein interaction network.

Figure 1

Culture and identification of Schwann cells. (A, D) The shape of NSCs and ASCs under an optical microscope, all cells were arranged in a long spindle shape and nucleus was oblong or ovoid. Scale bar: 200 μm (B, E, G) Adhesion assay of Schwann cells. Scale bar: 100 μm. (C, F) Immunofluorescent staining images of s100, DAPI, and Merge of NSCs and ASCs. (H) Proliferation assay of ASCs and NSCs. ^*P<0.05; ^**P<0.01; ^***P<0.001. Scale bar, 50 µm. NSCs, normal schwann cells; ASCs, activated schwann cells; OD, optical density; Au, absorbance units.

Figure 2

Expression signatures of differential methylation genes after PNI. (A) Concise experimental procedure for the Methylated DNA immunoprecipitation-sequencing. (B) Differential methylation genes were classified according their genomic architecture after PNI. (C) Chromosome distribution showed the numbers of regulated genes located at different chromosomes. (D) Heat map of nine aberrantly expressed genes. Vcl, vinculin; Bcar1, BCAR1 scaffold protein; Lamc1, laminin subunit γ1; Col5a3, collagen type V α3 chain; Col18a1, collagen type XVIII α1 chain; Ezr, ezrin; Itgb6, integrin subunit β6; Col3a1, collagen type III α1 chain; MeDIP-seq, methylated DNA immunoprecipitation-sequencing; PNI, peripheral nerve injury; ch, chromosome; NSC, normal Schwann cell; ASC, activated Schwann cell; qPCR, quantitative PCR.

Figure 3

Bioinformatics analysis. (A) GO and (B) KEGG pathway analysis of adhesion-associated differentially expressed genes in schwann cells. (C) ITGA/ITGB, ROCK, Fyn and Crk may be associated with cell proliferation and survival through the focal adhesion pathway. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; DMGs, differentially methylated genes; ECM, extracellular matrix.

Figure 4

Bioinformatics analysis and verification by RTq-PCR. (A) PPI network analysis of adhesion-associated differentially expressed genes in schwann cells. (B) The top 10 high-degree hub nodes in the PPI network. (C) Expression levels of adhesion-associated genes verified via reverse transcription-quantitative PCR. Values are expressed as the mean ± SEM. ^*P<0.05; ^**P<0.01; ^***P<0.001 vs. NSCs. PPI, protein-protein interaction; NSCs, normal Schwann cells; ASCs, activated Schwann cells; GO, Gene Ontology; Vcl, vinculin; Bcar1, BCAR1 scaffold protein; Lamc1, laminin subunit γ1; Col5a3, collagen type V α3 chain; Col18a1, collagen type XVIII α1 chain; Ezr, ezrin; Itgb6, integrin subunit β6; Col3a1, collagen type III α1 chain.