2022, Vol. 26 ›› Issue (23): 3620-3632

CRID3, a blocker of apoptosis-associated speck-like protein containing a card, influences local gene transcription in mice with acute spinal cord injury: a transcriptomic analysis

Li Jinglu1, 2, Wang Sainan1, 2, 3, Wang Yangyang1, 2, 3, Fu Guiqiang1, 2, 3, Wang Ying2, Hu Jianguo1, 2, Tang Jie1, 3, Lyu Hezuo1, 2, 3   

Abstract: BACKGROUND: Inflammasomes play an important role in spinal cord injury. Apoptosis-associated speck-like protein containing a CARD is a common adaptor protein of inflammasome. Our previous studies have shown that CRID3, a specific oligomerization blocker of apoptosis-associated speck-like protein containing a card, can inhibit the activation of inflammasome and the production of corresponding cytokines by inhibiting the oligomerization of apoptosis-associated speck-like protein containing a card, so as to improve the local microenvironment of the injured spinal cord and play a neuroprotective role. However, its effect on spinal cord injury at transcriptional level has not been reported.
OBJECTIVE: To investigate the effect of CRID3 on local gene transcription at acute phase (8 hours) of spinal cord injury in mice by using RNA-sequencing.  
METHODS: Thirty female C57BL/6 mice aged 8 weeks and weighing 18-20 g were divided into a sham operation group and a spinal cord injury group. Mice in the spinal cord injury group were randomly subdivided into a control group and a CRID3 administration group. Mice in the CRID3 administration group were intraperitoneally injected with CRID3 (50 mg/kg) after operation, while mice in the control group were injected with an equal volume of physiological saline solution. At 8 hours after spinal cord injury, three mice from each group were perfused, and the spinal cord was taken to make frozen sections that were then stained with hematoxylin-eosin to determine whether the spinal cord injury model was successfully established. Meanwhile, another three mice were selected from each group to taken spinal cord tissue. The total RNA was then extracted and purified for library preparation and RNA-sequencing. The differential gene expression of the three groups was analyzed by DESeq2 software. Another six mice were selected from the control group and the CRID3 administration group, and spinal cord tissue was taken to extract and purify total RNA. The results of RNA-sequencing were verified by real-time quantitative reverse transcription PCR. GOseq R and KOBAS software were used for gene ontology and Kyoto encyclopedia of genes and genomes enrichment analysis of the differentially expressed genes. Based on literature mining, signaling pathways related to inflammasomes were explored, and the effect of CRID3 on the expression level of related genes were further analyzed. String protein-protein interaction analysis was used to detect CRID3-related proteins.
RESULTS AND CONCLUSION: Hematoxylin-eosin staining results showed that the spinal cord injury model was successfully constructed. The results of RNA-sequencing showed that compared with the sham operation group, there were 5 661 differentially expressed genes in the control group, including 3 427 up-regulated genes and 2 224 down-regulated genes. Compared with the control group, 2 924 differentially expressed genes were found in the CRID3 administration group, including 1 409 up-regulated genes and 1 515 down-regulated genes. The results of gene ontology analysis showed that the differentially expressed genes were mainly rich in chemokine receptor binding, G-protein coupled receptor binding, extracellular-glutamate-gated ion channel activity, excitatory extracellular ligand-gated ion channel activity, transmembrane transporter activity, and acid phosphatase activity. Kyoto gene and genome encyclopedia analysis combined with literature mining showed that the signaling pathways related to inflammasome mainly included tumor necrosis factor, Toll-like receptor, nuclear factor‑κB, PI3K-Akt, hypoxia-inducible factor-1, MAPK, NOD-like receptor signaling pathways as well as pyrocytosis, leukocyte transendothelial migration, and interaction between cytokines and receptors. The genes most sensitive to CRID3 included Asc, Casp4, Cyba, Cybb, F11r, Hif1a, Il18, Il1b, Itgal, Itgam, Itgb2, Jam3, Mmp3, Mmp9, and Tlr4. The results of String protein-protein interaction analysis showed that inflammasome components (Nlrp1, Nlrp3, Nlrp6, Nlrc4, Aim2, ASC, Csap1, and Csap4) interacted closely, and they formed a complete link with interleubin 1b and interleubin 18 through Csap8. In addition, some important nodal proteins such as Actn1, Cxcl5, Cd14, Cyba, Cybb, Fgf2, Hif1a, F11r, Itgal, Itga2b, Itgam, Itgb1, Jam3, Mmmp3, and Tlr4 were identified. To conclude, after spinal cord injury, a large number of genes related to inflammasomes can be activated and the signaling pathways formed by these genes may be the cause or result of the activation of inflammasomes. CRID3 can directly or indirectly inhibit gene expression in the acute stage of spinal cord injury by inhibiting the expression of inflammasome-related genes. Its related molecules and signaling pathways are mainly related to inflammatory response, local hypoxia, cell adhesion, migration, differentiation, proliferation and apoptosis. CRID3 can improve the local microenvironment of spinal cord injury in the acute stage, and these key node molecules can also be used as new therapeutic intervention targets.

Key words: spinal cord injury, CRID3, mouse, RNA-sequencing, inflammasome