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    Role of circular RNA expression in the pathological progression after spinal cord injury
  • Figure 1|Establishment of SCI mouse models and expression profiles of circRNA.

    To explore expression profiles of circRNAs in the lesion epicenter after SCI, a mouse model of SCI was established using an Allen’s drop method. HE-stained sections of SCI and sham groups were observed by microscopy. Nuclei were stained dark blue by hematoxylin, while cytoplasm appeared pink and collagen fibers were pale pink after eosin staining. The spinal cord structure in sham groups was clear with intact and unruptured meninges, and a centered central tube. Moreover, there was no bleeding or signs of damage (Figure 1A). In contrast, the spinal cord structure of SCI groups exhibited significant damage and rupture, meningeal defects, and central tube offset deformation. Extensive red blood cell diffusion and inflammatory cell infiltration caused by vascular rupture were also observed (Figure 1B). A cluster map and volcano plot show differentially expressed circRNAs after TSCI (Figure 1C and D).

    Figure 2|Enriched GO terms and KEGG pathways of differentially expressed circRNAs in SCI mice. 

    To explore major biological functions and significantly enriched pathways, GO and KEGG analyses were performed. Differential expression profiles of circRNAs were further screened according to their expression level and log2(fold change). Enriched terms for differentially expressed genes identified by GO term analysis were used to determine their major biological functions. The results of GO enrichment analysis showed that the highest degree of enrichment was associated with ‘negative regulation of plasminogen activation’, ‘peptide cross linking’, ‘positive regulation of the endothelial cell apoptotic process’, and ‘macropinosome’ (Figure 2A). Analysis of significantly enriched KEGG pathways revealed the most important biochemical metabolic pathways and signal transduction pathways encoded by differentially expressed genes included ‘complement and coagulation cascades’, ‘glycerolipid metabolism’, ‘one carbon pool by folate’, ‘pentose phosphate pathway’, ‘bladder cancer’, and ‘P53 signaling pathway’ (Figure 2B). 

    Figure 3|circRNA–miRNA–mRNA regulatory interaction network analysis. 

    To investigate regulatory roles of the targeted interaction axis after SCI, we predicted the miRNA/circRNA axis using bioinformatic analyses. After TSCI, differential expression profiles of circRNAs and miRNAs were screened according to their expression and log2(fold change). miR-135b-5p was significantly downregulated (P < 0.05), as it satisfied the significance threshold of |log2(fold change)| ≥ 1. Indeed, miR-135b-5p showed the most significant reduction in expression after SCI (log2(fold change) = –1.01) (Figure 3A). High-throughput sequencing revealed significant upregulation (P < 0.05), of circAbca1 after TSCI (Figure 3B). Differentially expressed circRNAs were further screened based on their binding targets. In total, six differentially expressed circRNAs that potentially bind miR-135b-5p were screened, including circAbca1, circRNA5953, circRNA15386, circRNA3868, circRNA6769, and circRNA9429 (Figure 3C). Based on comprehensive analysis of backsplice junction loci, TargetScan, and miRanda scores, we identified circAbca1 as a key circRNA that interacts with miR-135b-5p to influence pathophysiological processes after TSCI. Reverse-screens identified miRNAs that bind to circAbca1, including miR-135b-5p, miR-23a-5p, miR-135a-5p, miR-155-5p, and miR-138-5p, which also target the bound mRNAs. Together with circAbca1, these miRNAs/mRNAs were used as the core for constructing the circRNA/miRNA interaction network after TSCI (Figure 3D).

    Figure 4|Validation of differential non-coding RNA expression and confirmation of relationships among non-coding RNAs.

    To verify the accuracy of high-throughput sequencing and confirm the circAbca1/miR-135b-5p relationship, we performed qRT-PCR and fluorescent reporter assays. qRT-PCR was used to validate expression of miR-135b-5p and circAbca1, which showed significant differential expression following library sequencing. qRT-PCR results were consistent with our sequencing results (Figure 4A and B). We predicted a targeted binding site for circAbca1 and miR-135b-5p (Figure 4C), and constructed a luciferase reporter plasmid (Figure 4D) to validate the targeted binding relationship. Luciferase activity of the miR-135b-5p mimic group was significantly lower than  that of the negative control group before mutation (P < 0.05), but this difference disappeared post-mutation. These results confirm that circAbca1 can bind to miR-135b-5p (Figure 4E), consistent with our predicted results.

    Figure 5|Validation of functional target gene expression and confirmation of the network relationship.

    To validate expression of Kruppel-like factor 4 (KLF4) and verify the miR-135b-5p/KLF4 relationship, qRT-PCR and fluorescent reporter assays were performed. Subsequently, the potential neuroinhibitory function of circAbca1 was determined by western blot assay. qRT-PCR was used to validate KLF4 expression, while high-throughput sequencing revealed significant upregulation (P < 0.05) of miR-135b-5p after TSCI (Figure 5A), consistent with our qRT-PCR results (Figure 5B). The Luc-KLF4 3′UTR-WT plasmid (Figure 5C) was synthesized to validate the targeted binding relationship. The binding site for KLF4 and miR-135b-5p was predicted by Targetscan software (Figure 5D). Luciferase reporter assay results indicated significantly decreased (P < 0.05) luciferase activity in 293T cells co-transfected with MT vector or miR?135b mimics (Figure 5E). 
    To confirm the correlation between circAbca1, miR-135b-5p, and KLF4 in primary mouse dorsal root ganglion spinal neurons, they were transfected with circAbca1 inhibitors. Transfection efficiency and expression of miR-135b and KLF4 were detected by qRT-PCR. The results revealed that silencing of circAbca1 increased miR-135b-5p expression, but decreased KLF4 expression (Figure 5F). To further explore the potential neuroprotective functions of circAbca1 inhibition, a western blot assay was conducted. The results show that GAP43 expression was upregulated after circAbca1 silencing (Figure 5G).

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  • 发布日期: 2021-03-20  浏览: 570
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