Comprehensive analysis of the differential expression profile of microRNAs in rats with spinal cord injury treated by electroacupuncture

Abstract

Abnormal microRNA (miRNA) expression has been implicated in spinal cord injury (SCI), but the underlying mechanisms are poorly understood. To observe the effect of electroacupuncture (EA) on miRNA expression profiles in SCI rats and investigate the potential mechanisms involved in this process, Sprague‑Dawley rats were divided into sham, SCI and SCI+EA groups (n=6 each). Basso, Beattie and Bresnahan (BBB) scoring and hematoxylin‑eosin staining of cortical tissues were used to evaluate spinal cord recovery with EA treatment 21 days post‑surgery across the three groups. To investigate miRNA expression profiles, 6 Sprague‑Dawley rats were randomly divided into SCI and SCI+EA groups (n=3 in each group) and examined using next‑generation sequencing. Integrated miRNA‑mRNA‑pathway network analysis was performed to elucidate the interaction network of the candidate miRNAs, their target genes and the involved pathways. Behavioral scores suggested that hindlimb motor functions improved with EA treatments. Apoptotic indices were lower in the SCI+EA group compared with the SCI group. It was also observed that 168 miRNAs were differentially expressed between the SCI and SCI+EA groups, with 29 upregulated and 139 downregulated miRNAs in the SCI+EA group. Changes in miRNA expression are involved in SCI physiopathology, including inflammation and apoptosis. Reverse transcription‑quantitative PCR measurement of the five candidate miRNAs, namely rno‑miR‑219a‑5p, rno‑miR‑486, rno‑miR‑136‑5p, rno‑miR‑128‑3p, and rno‑miR‑7b, was consistent with RNA sequencing data. Integrated miRNA‑mRNA‑pathway analysis suggested that the MAPK, Wnt and NF‑κB signaling pathways were involved in EA‑mediated recovery from SCI. The present study evaluated the miRNA expression profiles involved in EA‑treated SCI rats and demonstrated the potential mechanism and functional role of miRNAs in SCI in rats.

Keywords: next-generation sequencing; microrna profile; spinal cord injury; electroacupuncture; reverse transcription-qPcr.

Figure 1.

Effect of EA on the motor function of SCI rats. Results of the BBB test in sham, SCI and SCI+EA-treated groups at 1, 3, 7 and 21 days following SCI surgery. The BBB score was significantly lower in the SCI group compared with the sham and SCI+EA groups at 3, 7 and 21 days. Data are presented as the mean ± SD. BBB, Basso, Beattie, and Bresnahan; SCI, spinal cord injury; EA, electroacupuncture. n=6 in each group. *P<0.05 SCI vs. sham; ^#P<0.05 SCI vs. SCI+EA; ^{^}P<0.05 are shown for SCI+EA vs. sham.

Figure 2.

Hematoxylin-eosin staining of SCI rat tissues. Representative morphological photographs under light microscopy after 12 rats with SCI were divided into untreated and EA-treated groups, vs. 6 healthy rats. (A) Sham group. (B) SCI group. (C) SCI+EA group. Black, red and white arrows represent nerve cells, inflammatory cells and tissue vacuoles, respectively. The number of black arrows in sham group and SCI+EA group was more than in SCI group. The number of red and white arrows in SCI group was more than that in sham group and SCI+EA group; n=6 in each group; magnification, ×200. Scale bar=100 µm. SCI, spinal cord injury; EA, electroacupuncture.

Figure 3.

Detection of tissue inflammatory factors. (A) TNF-α and (B) IL-1β levels were detected by ELISA. (C) The relative expression levels of TNF-α and IL-1β were detected by reverse transcription-quantitative PCR. *P<0.05. SCI, spinal cord injury; EA, electroacupuncture; TNF-α, tumor necrosis factor-α; IL, interleukin.

Figure 4.

Differential expression of spinal cord tissue miRNAs between rats in the SCI and SCI+EA groups. (A) Heatmap depicting the expression of the differentially expressed miRNAs with upregulated (red) and downregulated (green). Samples and miRNAs have been reordered by hierarchical clustering. (B) Top 20 up- or downregulated differentially expressed miRNAs between SCI+EA and SCI group rats based on miRNA sequencing. SCI, spinal cord injury; EA, electroacupuncture; miRNA, micro RNA.

Figure 5.

GO and KEGG analyses of the target genes of differentially expressed miRNAs. (A) GO analysis of the significantly enriched target genes. The top 20 significantly enriched items are listed. The degree of GO enrichment was determined by P-value, enrichment factor and miRNA target numbers. Dot sizes and colors indicate the number of target genes and -log(P-value), respectively. (B) Pathway enrichment analysis for miRNA targets through KEGG. Enrichment scores are presented as -log(P-value). GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; miRNA, micro RNA.

Figure 6.

Validation of candidate differentially expressed miRNAs by RT-qPCR. Bar graph representing expression levels of 5 miRNAs identified by next-generation sequencing. Data are presented as the mean ± SD of relative expression levels measured by RT-qPCR. n=3. *P<0.05. SCI, spinal cord injury; EA, electroacupuncture; miRNA, micro RNA; RT-qPCR, reverse transcription-quantitative PCR.

Figure 7.

Target gene analysis of candidate miRNAs. (A) Predicted miRNA-mRNA-pathway network. miRNAs presented in green are downregulated, while those in red are upregulated in the SCI+EA group, vs. the SCI group. Target genes are represented by yellow hexagons. Blue diamonds represent signaling pathways. (B) Transfection efficiency of miR-136-5p mimics was verified by reverse transcription-quantitative PCR. (C) mRNA expression level of Sema3a, Vangl2 and Gng3 in BV2 microglial cells. Data are presented as the mean ± SD. n=3 in each group. *P<0.05 and ****P<0.0001. SCI, spinal cord injury; EA, electroacupuncture; miR/miRNA, micro RNA; Sema3a, semaphoring 3A; Vangl2, Van Gogh Drosophila-like planar cell polarity protein 2; Gng3, G protein subunit γ 3.

Figure 8.

Effects of miR-136-5p on cell proliferation and apoptosis in BV2 microglial cells. (A) BV2 microglial cells were transfected with miR-136-5p mimics, and cell proliferation was quantified by administering Cell Counting Kit-8 after 0, 24, 48, 72 or 96 h of incubation with miR-136-5p mimics or NC mimics. OD was measured at 450 nm after 1 h. Data are presented as the mean ± SD. (B) Annexin V/PI double-staining method to detect microglial cell apoptosis in the NC mimic and miR-136-5p mimic groups. n=6 in each group. *P<0.05 and ***P<0.001 miR-136-5p mimics group vs. NC mimics group. OD, optical density; NC, negative control; PI, propidium iodide; FITC, fluorescein isothiocyanate.