Analyses of long non-coding RNA and mRNA profiles in the spinal cord of rats using RNA sequencing during the progression of neuropathic pain in an SNI model

Abstract

The pathogenesis of neuropathic pain (NP) is characterized by an increased responsiveness of nociceptive neurons in the nervous system. However, the molecular mechanisms underpinning the NP still remain elusive. Recent data suggest that long non-coding RNAs (lncRNAs) regulate expression of NP-associated genes. Herein, we analyzed lncRNAs and mRNA profiles in the spinal cord of rats by RNA sequencing during the progression of NP in a spared nerve injury (SNI) model. Sprague-Dawley (SD) rats were employed for the establishment of the SNI models, and nociceptive responses to mechanical and thermal stimuli were measured 3 hours prior to surgery and on postoperative days 1, 3, 7 and 14, with L_4-5 spinal cords extracted from three SD rats under deep anesthesia at each time point after behavioral test. SNI rats exhibited higher sensitivity to mechanical stimuli from days 1 to 14. Mechanical hyperalgesia reached a steady peak at day14 after surgery, whereas thermal allodynia did not develop. The results of second-generation sequencing suggested that the expression profiles of lncRNAs and mRNAs were significantly altered in spinal cords of SNI rats versus the control rats at different stages during NP. Differentially expressed (DE) lncRNAs and mRNAs were demonstrated at each stage during the NP course using Volcano Plot, Venn and Hcluster heatmap analyses. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway analyses were performed to predict the functionalities of differentially expressed lncRNAs and target genes. Protein interaction networks were constructed based on the correlation analyses of DE lncRNA target proteins at 7 and 14 days after SNI, respectively. Taken together, our results revealed the profiles of lncRNAs and mRNAs in the rat spinal cord under an NP condition. These lncRNAs and mRNAs may represent new therapeutic targets for the treatment of NP.

Keywords: LncRNA; Neuropathic pain; Sequencing Data Analysis; Spared nerve injury; Spinal cord.

Figure 1.

Nociceptive behavior developed in SNI model rats. MWT (mechanical withdrawal threshold) in each time points (A), TWL (thermal withdrawal latency) in each time points (B). n = 18, *p < 0.001 compared T₀.

Figure 2.

The expression profiling changes of lncRNAs during the different stages of SNI. Vocalno Plot indicated up-regulated and down-regulated lncRNAs at different time points of SNI model (Fig. 2A); Venn diagram showed the number of overlap lncRNAs during the different stages of SNI in Fig. 2B; Heat map of lncRNAs showed hierarchical clustering of changed lncRNAs at the different time points. In clustering analysis, up-regulated and down-regulated genes are colored in red and blue, respectively. The sequencing samples at 0d, 1d, 3d, 7d and 2w were labeled d0, d1, d3, d7 and w2, respectively; the serial numbers of samples in each time point were labeled ending (Fig. 2C).

Figure 3.

The expression profiling changes of mRNAs during the different stages of SNI. Vocalno Plot indicated up-regulated and down-regulated mRNAs at different time points of SNI model (Fig. 3A); Venn diagram showed the number of overlap mRNAs during the different stages of SNI in Fig. 3B; Heat map of mRNAs showed hierarchical clustering of changed mRNAs at the different time points. In clustering analysis, up-regulated and down-regulated genes are colored in red and blue, respectively. The sequencing sample at 0d, 1d, 3d, 7d and 2w were labeled d0, d1, d3, d7 and w2, respectively; the serial numbers of samples in each time point were labeled ending (Fig. 3C).

Figure 4.

QPCR validations of seven regulated lncRNAs in the spinal cord from SNI rats. The expressions of lncRNAs(A) were significantly up-regulated at 7 days after SNI. The expressions of lncRNAs(B) were significantly down-regulated at 7 days after SNI. One-way ANOVA followed by Tukey's multiple comparison test. ***P < 0.001.

Figure 5.

Enriched GO Terms with SNI pathogenesis. The significant molecular function, biological process and cellular component changed mRNAs in different stages of SNI. SNI 1d vs. 0d (Fig. 5A), SNI 3d vs. 0d (Fig. 5B), SNI 7d vs. 0d (Fig. 5C) and SNI 2w vs. 0d (Fig. 5D).

Figure 6.

DE lncRNAs co-localized genes enriched KEGG pathway scatterplot with SNI pathogenesis. LncRNAs enriched KEGG pathway scatterplot showing statistics of pathway enrichment at different time points. Pathway of lncRNAs co-localized genes at different stage of SNI including SNI 1d vs. 0d (Fig. 6A), SNI 3d vs. 0d (Fig. 6B), SNI 7d vs. 0d (Fig. 6C) and SNI 2w vs. 0d (Fig. 6D).

Figure 7.

DE lncRNAs co-expressed genes enriched KEGG pathway scatterplot with SNI pathogenesis. LncRNAs enriched KEGG pathway scatterplot showing statistics of pathway enrichment at different time points. Pathway of lncRNAs co-expressed genes at different stage of SNI including SNI 1d vs. 0d (Fig. 7A), SNI 3d vs. 0d (Fig. 7B), SNI 7d vs. 0d (Fig. 7C) and SNI 2w vs. 0d (Fig. 7D).

Figure 8.

DE mRNAs enriched KEGG pathway scatterplot with SNI pathogenesis. LncRNAs enriched KEGG pathway scatterplot showing statistics of pathway enrichment at different time points. Pathway of lncRNAs co-expressed genes at different stages of SNI including SNI 1d vs. 0d (Fig. 8A), SNI 3d vs. 0d (Fig. 8B), SNI 7d vs. 0d (Fig. 8C) and SNI 2w vs. 0d (Fig. 8D).

Figure 9.

Protein interaction network analysis of DE mRNA in rats at 7d after SNI.

Figure 10.

Protein interaction network analysis of DE mRNA in rats at 14d after SNI.