Chronic constriction injury of sciatic nerve changes circular RNA expression in rat spinal dorsal horn

Abstract

Background: Mechanisms of neuropathic pain are still largely unknown. Molecular changes in spinal dorsal horn may contribute to the initiation and development of neuropathic pain. Circular RNAs (circRNAs) have been identified as microRNA sponges and involved in various biological processes, but whether their expression profile changes in neuropathic pain condition is not reported.

Methods: To test whether neuropathic pain influences circRNA expression, we developed a sciatic chronic constriction injury (CCI) model in rats. The CCI ipsilateral spinal dorsal horns of lumbar enlargement segments (L3-L5) were collected, and the total RNA was extracted and subjected to Arraystar Rat circRNA Microarray. Quantitative real-time polymerase chain reaction (qPCR) was used to confirm the circRNA expression profile. To estimate functions of differential circRNAs, bioinformatics analyses including gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes Pathway analyses were performed for the top 100 circRNAs and circRNA-microRNA networks were constructed for the top 10 circRNAs.

Results: circRNA microarrays showed that 469 circRNAs were differentially expressed between CCI and sham-operated rats (fold change ≥2). In all, 363 of them were significantly upregulated, and the other 106 were downregulated in the CCI group. Three of them (circRNA_013779, circRNA_008008, and circRNA_003724) overexpressed >10 times after CCI insult. Expression levels of eight circRNAs were verified using qPCR. GO analysis revealed that thousands of predicted target genes were involved in the biological processes, cellular component, and molecular function; in addition, dozens of these genes were enriched in the Hippo signaling pathway, MAPK signaling pathway, and so on. Competing endogenous RNAs analysis showed that circRNA_008008 and circRNA_013779 are the two largest nodes in the circRNA-microRNA interaction network of the top 10 circRNAs.

Conclusion: CCI resulted in a comprehensive expression profile of circRNAs in the spinal dorsal horn in rats. CircRNAs in the dorsal horn could be helpful to reveal molecular mechanisms of neuropathic pain.

Keywords: circRNA–microRNA interaction; circular RNA; microarray; neuropathic pain.

Figure 1

CCI-induced mechanical hypersensitivity. Notes: Changes in MWT were assessed with the electronic von Frey filament on ipsilateral hind paws of CCI and sham-operated rats. n=6 in both groups. All data are represented as mean ± SD. Statistical analyses consisted of repeated measures two-way ANOVA tests. ***P<0.001 between sham and CCI groups. Abbreviations: CCI, chronic constriction injury; SD, standard deviation; ANOVA, analysis of variance; MWT, mechanical withdrawal thresholds.

Figure 2

CircRNA expression between the CCI and sham-operated rats. Notes: (A) Hierarchical clustering shows differential circRNA expression profiles between two groups. The upregulated circRNAs in the CCI group are shown in red, and the downregulated circRNAs are indicated in green. (B) Box plots show the distribution of circRNAs for the two compared samples. The distributions were nearly the same after normalization. (C) Scatter plots assess the circRNA expression differences between the two compared groups. The circRNAs above the top green line and below the bottom green line indicated differential circRNAs (fold change ≥2 or ≤−2, respectively). Abbreviations: CCI, chronic constriction injury; circRNA, circular RNA.

Figure 3

Validation of eight differentially expressed circRNAs by qPCR. Notes: qPCR results were calculated with 2−ΔΔCt method and normalized to GAPDH expression in the sham and CCI group. Bars represent the mean ± SD; positive fold change means overexpression in the CCI group. Each qPCR assay was performed three times. Abbreviations: CCI, chronic constriction injury; SD, standard deviation; circRNA, circular RNA; qPCR, quantitative real-time polymerase chain reaction.

Figure 4

GO enrichment analysis. GO enrichment corresponds to the top 100 circRNAs. Abbreviations: circRNAs, circular RNAs; GO, gene ontology.

Figure 5

Top 20 KEGG pathways with the highest enrichment score. Notes: Pathways correspond to the top 100 circRNAs. The vertical axis is the pathway category and the horizontal axis is the –LgP of the pathway; LgP is the logarithm of the P-value, and P<0.05 was considered significant. Abbreviations: circRNAs, circular RNAs; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 6

CircRNA–microRNA (ceRNA) network. Notes: The circRNA–microRNA ceRNA network was drawn with the Cytoscape software. The size of each green node represents the functional connectivity of each circRNA. Rno_circRNA_008008 and rno_circRNA_013779 are the two largest nodes in the network. Abbreviations: circRNAs, circular RNAs; ceRNA, competing endogenous RNAs.