Underlying Mechanism of the circ_0011462/miRNA-1200/SP1 Axis in Regulating Inflammatory Response, Apoptosis, and Neural Regeneration in a Spinal Cord Injury Model

Objective 

To investigate the regulatory mechanism of a circular RNA (circRNA), circ_001146, on the miR-1200/SP1 axis in spinal cord injury (SCI).

Methods 

In the animal model, male Sprague–Dawley rats of 8 weeks old, weighing 220-250 g, were randomly assigned to 5 groups (n = 5 per group), including the sham-operated (SHAM) group, SCI group, circ_0011462 knockdown (sh-circ_0011462) group, circ_0011462 knockdown + empty plasmid control (NC) group, and circ_0011462 knockdown + SP1 overexpression (overexpression-SP1) group. Motor function recovery of the rats in different groups was evaluated at multiple time points using the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale, tail-flick latency (TFL) test, and grid walking test. Nissl staining was performed to measure the number of motor neurons in the anterior horn of the spinal cord. Immunohistochemistry assays were performed to determine the expression of pNF and synaptophysin in the spinal tissue. The quantity of M1-type microglia was assessed using multiplex immunofluorescence. Enzyme-linked immunosorbent assay (ELISA) was performed to measure the levels of IL-1β, IL-6, and TNF-α in peripheral blood across the groups. In addition, cellular experiments included qPCR to determine the mRNA levels of circ_001146, miR-1200, and SP1 in different groups, Western blot (WB) to detect the protein levels of SP1, BCL-2, and Caspase-3, flow cytometry to assess cell apoptosis rates, and phase-contrast microscopy to observe cell morphological changes.

Results 

In the animal experiment, at 21 and 28 days post surgery, the sh-circ_0011462 group exhibited significantly higher BBB scores (7.00 ± 1.20 and 10.00 ± 2.21) and TFL scores (1.21 ± 0.41 and 1.19 ± 0.31) compared with the SP1 overexpression group (BBB: 5.00 ± 0.60 and 6.00 ± 0.80; TFL: 0.80 ± 0.25 and 0.70 ± 0.20) and the SCI group (BBB: 4.00 ± 0.60 and 5.00 ± 0.70; TFL: 0.60 ± 0.21 and 0.50 ± 0.18) (all P < 0.05). In contrast, Gridwalk scores in the sh-circ_0011462 group (1.38 ± 0.31 and 1.22 ± 0.33) were significantly lower than those in the overexpression-SP1 group (2.32 ± 0.37 and 2.40 ± 0.41) and the SCI group (2.72 ± 0.45 and 2.80 ± 0.42) (P < 0.05 for all comparisons). At day 28 post-surgery, the levels of IL-1β, IL-6, and TNF-α in the sham group were lower than those in all other groups (P < 0.05). The SCI and overexpression-SP1 groups showed elevated levels of these cytokines, all of which were significantly higher than those in the sh-circ_0011462 group (all P < 0.05). Immunostaining revealed that the number of iNOS-positive cells in the sh-circ_0011462 group was significantly lower than that in the SCI group and the overexpression-SP1 group (P < 0.05). Moreover, the number of motor neurons, axonal length, and synapse density in the anterior horn of rats in the sh-circ_0011462 group were all significantly greater than those in the SCI and overexpression-SP1 groups (all P < 0.05). In cellular experiments, qPCR and Western blot assays showed that circ_0011462 knockdown resulted in increased expression of miR-1200, decreased mRNA and protein levels of SP1, downregulation of Caspase-3, upregulation of Bcl-2, reduced apoptosis rate and enhanced axonal growth in PC12 cells. These effects were reversed by transfection with the miR-1200 inhibitor or the overexpression-SP1 plasmid. Transfection with the miR-1200 mimic produced effects similar to those of circ_0011462 knockdown, while co-transfection with the SP1 plasmid attenuated these effects. Flow cytometry further confirmed that apoptosis rates in PC12 cells treated with H₂O₂ or NC vectors were significantly higher than those in the control group (all P < 0.05). In the sh-circ_0011462 group, apoptosis rates significantly decreased (P < 0.05). When the miR-1200 inhibitor or the SP1 plasmid was transfected into the sh-circ_0011462 group, apoptosis rates increased again (P < 0.05). Moreover, co-transfection with SP1 plasmid increased the apoptosis rate, which was higher than that in the pcDNA vector control group (P < 0.05). Dual-luciferase reporter assays confirmed that miR-1200 directly targeted circ_0011462 and SP1. Compared with the miR-NC group, miR-1200 mimic significantly reduced the luciferase activity of circ_0011462-wt and SP1-wt (P < 0.05), while no significant changes were observed in their respective mutant constructs (P > 0.05).

Conclusion 

circ_001146 may target and modulate the miR-1200/SP1 axis to ameliorate the pathological conditions of SCI, thereby reducing inflammatory responses, inhibiting neuronal apoptosis, and promoting neural regeneration.

 

Keywords: circ_001146, miR-1200, SP1, Spinal cord injury

 

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