Identification of key microRNAs and the underlying molecular mechanism in spinal cord ischemia-reperfusion injury in rats

Abstract

Spinal cord ischemia-reperfusion injury (SCII) is a pathological process with severe complications such as paraplegia and paralysis. Aberrant miRNA expression is involved in the development of SCII. Differences in the experimenters, filtering conditions, control selection, and sequencing platform may lead to different miRNA expression results. This study systematically analyzes the available SCII miRNA expression data to explore the key differently expressed miRNAs (DEmiRNAs) and the underlying molecular mechanism in SCII. A systematic bioinformatics analysis was performed on 23 representative rat SCII miRNA datasets from PubMed. The target genes of key DEmiRNAs were predicted on miRDB. The DAVID and TFactS databases were utilized for functional enrichment and transcription factor binding analyses. In this study, 19 key DEmiRNAs involved in SCII were identified, 9 of which were upregulated (miR-144-3p, miR-3568, miR-204, miR-30c, miR-34c-3p, miR-155-3p, miR-200b, miR-463, and miR-760-5p) and 10 downregulated (miR-28-5p, miR-21-5p, miR-702-3p, miR-291a-3p, miR-199a-3p, miR-352, miR-743b-3p, miR-125b-2-3p, miR-129-1-3p, and miR-136). KEGG enrichment analysis on the target genes of the upregulated DEmiRNAs revealed that the involved pathways were mainly the cGMP-PKG and cAMP signaling pathways. KEGG enrichment analysis on the target genes of the downregulated DEmiRNAs revealed that the involved pathways were mainly the Chemokine and MAPK signaling pathways. GO enrichment analysis indicated that the target genes of the upregulated DEmiRNAs were markedly enriched in biological processes such as brain development and the positive regulation of transcription from RNA polymerase II promoter. Target genes of the downregulated DEmiRNAs were mainly enriched in biological processes such as intracellular signal transduction and negative regulation of cell proliferation. According to the transcription factor analysis, the four transcription factors, including SP1, GLI1, GLI2, and FOXO3, had important regulatory effects on the target genes of the key DEmiRNAs. Among the upregulated DEmiRNAs, miR-3568 was especially interesting. While SCII causes severe neurological deficits of lower extremities, the anti-miRNA oligonucleotides (AMOs) of miR-3568 improve neurological function. Cleaved caspase-3 and Bax was markedly upregulated in SCII comparing to the sham group, and miR-3568 AMO reduced the upregulation. Bcl-2 expression levels showed a opposite trend as cleaved caspase-3. The expression of GATA6, GATA4, and RBPJ decreased after SCII and miR-3568 AMO attenuated this upregulation. In conclusion, 19 significant DEmiRNAs in the pathogenesis of SCII were identified, and the underlying molecular mechanisms were validated. The DEmiRNAs could serve as potential intervention targets for SCII. Moreover, inhibition of miR-3568 preserved hind limb function after SCII by reducing apoptosis, possibly through regulating GATA6, GATA4, and RBPJ in SCII.

Keywords: Bioinformatics analysis; Spinal cord ischemia reperfusion injury; Transcription factor analysis; microRNA.

Figure 1. The analysis process of the study. SCII, spinal cord ischemia-reperfusion injury; DEmiRNAs, differentially expressed miRNAs.

Figure 2. Distribution of DEmiRNAs in 23 SCIIs.

(A) Upregulation of the proportion of DEmiRNAs in the SCII datasets. A total of 72 upregulated miRNAs were obtained. The number of DEmiRNAs supported by one dataset, two datasets, and three datasets was 51, nine (including an inconsistent expression miRNA: miR-22-3p), and 1. (B) Downregulation of the proportion of DEmiRNAs in the SCII datasets. A total of 79 downregulated miRNAs were obtained. The number of DEmiRNAs supported by one dataset, two datasets, and three datasets was 58, 9, and one.

Figure 3. Expression of key DEmiRNAs following SCII.

(A) The expression of key upregulated DEmiRNAs following SCII. (B) The expression of key downregulated DEmiRNAs following SCII. n = 4 for per group. Data were analyzed with Student’s t-test. *p < 0.05, versus the sham group.

Figure 4. The KEGG pathway analysis and the GO annotations for biological process of top 10 most significant enrichment terms for target genes of DEmiRNAs.

(A) KEGG pathway analysis for upregulation DEmiRNAs target genes. (B) KEGG pathway analysis for downregulation DEmiRNAs target genes. (C) The biological process of GO annotations for upregulation DEmiRNAs target genes. (D) The biological process of GO annotations for downregulation DEmiRNAs target genes.

Figure 5. Transcription factor analysis of key DEmiRNAs target genes in SCII.

(A) The Venn Diagram of transcription factors of DEmiRNAs target genes. The purple-blue background represents the number of transcription factor analysis for upregulated DEmiRNAs target genes. The pink background represents the number of transcription factor analysis for downregulated DEmiRNAs target genes. (B) The transcription factor-DEmiRNAs target genes regulation network. The redtriangle represents the transcription factor; the purple hexagon represents upregulated DEmiRNAs target genes; the green hexagon represents downregulated DEmiRNAs target genes; the orange hexagon represents target genes of both upregulated and downregulated DEmiRNAs.

Figure 6. A miR-3568-biological processes-gene network.

Figure 7. Effects of AMO-3568 following SCII.

Effects of AMO-3568 following SCII. (A) Tarlov scores. n = 8 for per group. (B–E) The protein expression levels of cleaved caspase-3, Bax, and Bcl-2 were measured with Western blot assay. n = 4 for per group. Data were analyzed with the one-way ANOVA followed by the Tukey’s test. *p < 0.05, versus the sham group, ^#p < 0.05 versus the SCII group.

Figure 8. Effects of AMO-3568 on the protein expression levels of RPBJ (A, B), GATA6 (A, C) and GATA4 (A, D) were measured by Western blot assay.

n = 4 for per group. Data were analyzed by one-way ANOVA followed by the Tukey’s test. *p < 0.05, versus the sham group, ^#p < 0.05 versus the SCII group.