Identification and coregulation pattern analysis of long noncoding RNAs following subacute spinal cord injury

Abstract

Long noncoding RNAs (lncRNAs) have been demonstrated to play critical regulatory roles in posttranscriptional and transcriptional regulation in eukaryotic cells. However, the characteristics of many lncRNAs, particularly their expression patterns in the lesion epicenter of spinal tissues following subacute spinal cord injury (SCI), remain unclear. In this study, we determined the expression profiles of lncRNAs in the lesion epicenter of spinal tissues after traumatic SCI and predicted latent regulatory networks. Standard Allen's drop surgery was conducted on mice, and hematoxylin and eosin staining was used to observe the damaged area. High-throughput sequencing was performed to identify the differential expression profiles of lncRNAs. Quantitative real-time polymerase chain reaction was conducted to evaluate the quality of the sequencing results. Bioinformatics analyses, including Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis, coexpression analysis, and protein-protein interaction analysis, were performed. Targeted binding of lncRNA-miRNA-mRNA was predicted by TargetScan and miRanda. A total of 230 differentially expressed lncRNAs were identified and preliminarily verified, and some potential regulatory networks were constructed. These findings improve our understanding of the mechanisms underlying subacute SCI; differentially expressed lncRNAs are closely involved in pathophysiological processes by regulating multiple pathways. Further studies are essential for revealing the exact mechanism underlying competing endogenous RNA pathways in vivo and in vitro.

Keywords: bioinformatics; long noncoding RNA; regulatory network; subacute spinal cord injury.

Figure 1

Hematoxylin and eosin staining of spinal cord samples, Sham group (A), 1 day after spinal cord injury (B), and 7 days after spinal cord injury(C) (Scar bars in low‐power field =200 µm, Scar bar in high‐power field = 100 µm)

Figure 2

Overview of the analysis pipeline. lncRNA, long noncoding RNA; mRNA, messenger RNA; miRNA, microRNA

Figure 3

Genome Mapping and Genome‐wide identification. Distribution of lncRNAs in the genome of different samples. There are six layers from the outside to the inside, and each layer shows the chromosome distribution and expression of lncRNAs of one sample. The outer three layers represent the SCI group, and the inner three layers represent the Sham group. The height of the column in each layer represents the expression level, and the higher the expression level, the higher the column height (A). RNA distribution of six types of lncRNAs along each chromosome. Known lncRNAs (class code = , depicted in orange), intronic lncRNAs (class code i, depicted in light green), lncRNAs sharing a reference with at least 1 splice junction (class code j, depicted in the dark green), lncRNA of generic exonic overlap with a reference transcript (class code o, depicted in blue), intergenic lncRNA (class code u, depicted in violet), and antisense lncRNA (class code x, depicted in pink) are presented in physical bins of 500 kb for each chromosome (B). Heatmap of top differentially expressed lncRNAs (C). Barplot of DE lncRNAs (D). Volcano plots showing variance in differentially expressed lncRNA (E). Red and blue points indicate up‐ and downregulated lncRNAs, respectively. DE, differentially expressed; lncRNA, long noncoding RNA

Figure 4

Validation of differential lncRNA, miRNA, and mRNA expression. Sequencing results of lncRNAs (A), miRNAs (C), and mRNA (E); qRT‐PCR validation of putative lncRNAs (B), miRNAs (D), and mRNAs (F). mean ± SD, n = 3, **p < 0.005, ***p < 0.001 (two sample t‐test). lncRNA, long noncoding RNA; mRNA, messenger RNA; miRNA, microRNA; qRT‐PCR, real‐time quantitative polymerase chain reaction

Figure 5

Basic property comparison of lncRNAs and mRNAs. Exon numbers of lncRNAs and mRNAs (A). Transcript lengths of lncRNAs and mRNAs (B). ORF lengths of lncRNAs and mRNAs (C) and (D). Expression levels of lncRNAs and mRNAs, mean ± SD (E). lncRNA, long noncoding RNA; mRNA, messenger RNA; miRNA, microRNA; ORF, open reading frames; SD, standard deviation

Figure 6

Interaction protein–protein network analysis of differentially expressed genes

Figure 7

Enriched GO terms (A) and KEGG pathways (B) of differentially expressed lncRNA‐related transcripts. The rich factor is the ratio of the number of different genes to the total number of genes in the database; a higher rich factor value indicates a greater enrichment degree. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes

Figure 8

Coexpression network analyses of top five most differentially expressed transcripts (A). Enriched GO terms (B) and KEGG pathways (C) of differentially expressed lncRNAs coexpressed transcripts. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; lncRNA, long noncoding RNA

Figure 9

LncRNA–miRNA–mRNA regulatory interaction network analysis of top 10 most differentially expressed lncRNAs. lncRNA, long noncoding RNA; mRNA, messenger RNA; miRNA, microRNA