Upregulation of miR-199a-5p Protects Spinal Cord Against Ischemia/Reperfusion-Induced Injury via Downregulation of ECE1 in Rat

Abstract

Ischemia-reperfusion (I/R)-induced spinal cord injury can cause apoptotic damage and subsequently act as a blood-spinal cord barrier damage. MicroRNAs (miRNAs) contributed to the process of I/R injury by regulating their target mRNAs. miR-199a-5p is involved in brain and heart I/R injury; however, its function in the spinal cord is not yet completely clarified. In this study, we investigated the role of miR-199a-5p on spinal cord I/R via the endothelin-converting enzyme 1, especially the apoptosis pathway. In the current study, the rat spinal cord I/R injury model was established, and the Basso Beattie Bresnahan scoring, Evans blue staining, HE staining, and TUNEL assay were used to assess the I/R-induced spinal cord injury. The differentially expressed miRNAs were screened using microarray. miR-199a-5p was selected by unsupervised hierarchical clustering analysis. The dual-luciferase reporter assay was used for detecting the regulatory effects of miR-199a-5p on ECE1. In addition, neuron expression was detected by immunostaining assay, while the expressions of p-ERK, ERK, p-JNK, JNK, caspase-9, Bcl-2, and ECE1 were evaluated by Western blot. The results indicated the successful establishment of the I/R-induced spinal cord injury model; the I/R induced the damage to the lower limb motor. Furthermore, 18 differentially expressed miRNAs were detected in the I/R group compared to the sham group, and miR-199a-5p protected the rat spinal cord injury after I/R. Moreover, miR-199a-5p negatively regulated ECE1, and silencing the ECE1 gene also protected the rat spinal cord injury after I/R. miR-199a-5p or silencing of ECE1 also regulated the expressions of caspase-9, Bcl-2, p-JNK, p-ERK, and ECE1 in rat spinal cord injury after I/R. Therefore, we demonstrated that miR-199a-5p might protect the spinal cord against I/R-induced injury by negatively regulating the ECE1, which could aid in developing new therapeutic strategies for I/R-induced spinal cord injury.

Keywords: Apoptosis; Blood–spinal cord barrier; ECE1; Ischemia/reperfusion; MiR-199a-5p.

Fig. 1

I/R-induced damage of lower limb motor. The rat spinal cord I/R injury model was established. a The lower limb motor function was assessed using BBB scoring criteria at 0, 6, 12, 24, 48, and 72 h post-injury. b EB fluorescence, number of neurons, and cell apoptosis were detected by EB, H&E, and TUNEL assays at 24 h post-injury, respectively. c EB fluorescence density was quantitatively analyzed by EB assay. d The number of neurons was counted according to H&E staining. e The rate of apoptosis was calculated according to the TUNEL assay

Fig. 2

MiRNAs expression in the rat spinal cord after I/R. The rat spinal cord I/R injury model was established. a Heat map of the differentially expressed miRNAs in the sham (n = 6) and I/R groups (n = 6). Each column indicates the expression profile of a sample; and red is high expression, green is low expression. b miR-199a-5p expression was analyzed by qRT-PCR assay in the sham and model groups

Fig. 3

Effects of miR-199a-5p on the rat spinal cord injury after I/R. The rat spinal cord I/R injury model was established and transfected with negative control (NC), miR-199a-5p mimic, and miR-199a-5p inhibitor, respectively. a The mRNA expression level of miR-199a-5p was detected by qRT-PCR assay. b The lower limb motor function of rats was analyzed by BBB scoring criteria at spinal cord I/R injury at 0, 6, 12, 24, 48, and 72 h post-reperfusion. c EB fluorescence, number of neurons, and cell apoptosis were measured by EB, H&E, and TUNEL assays at 24 h post-injury, respectively. d EB fluorescence density, e number of neurons, and f cell apoptosis were analyzed quantitatively. g The protein expression levels of p-ERK, ERK, p-JNK, JNK, caspase-9, Bcl-2, and ECE1 were measured by Western blot assay. h The putative complementary site of miR-199a-5p and ECE1 was obtained based on TargetScan prediction. Cells were transfected with wild-type ECE1 and mutant-type ECE1, and NC or miR-199a-5p mimic, respectively. The luciferase intensity was measured by dual-luciferase reporter assay. **P < 0.01 vs. NC group. a–c P < 0.05 vs. sham, I/R and NC, respectively

Fig. 4

miR-199a-5p inducing neuron proliferation in I/R-induced spinal cord injury rats. The rat spinal cord I/R injury model was established and transfected with negative control (NC), miR-199a-5p mimic, and miR-199a-5p inhibitor, respectively. a The immunostaining assay was performed to assess the expression of neuron-specific beta III tubulin. b Neuron-specific beta III tubulin expression was quantified. DAPI indicates the nucleus. a–c P < 0.05 vs. sham, I/R and NC, respectively

Fig. 5

Silencing of ECE1 exerting a protective effect on the rat spinal cord injury after I/R. The rat spinal cord I/R injury model was established and transfected with negative control (NC) and shRNA-ECE1s, respectively. (a) The mRNA expression level of ECE1 was accessed by qRT-PCR assay. b The protein expression level of ECE1 was analyzed by Western blot assay. c The lower limb motor function of rats was evaluated by BBB scoring criteria of spinal cord I/R injury at 0, 6, 12, 24, 48, and 72 h post-reperfusions. d EB fluorescence, number of neurons, and cell apoptosis were measured by EB, H&E, and TUNEL assays at 24 h post-injury, respectively. e EB fluorescence density, f number of neurons, and g cell apoptosis were analyzed quantitatively, respectively. h Expressions of p-ERK, ERK, p-JNK, JNK, caspase-9, Bcl-2, and ECE1 were measured by Western blot assay. a–c P < 0.05 vs. sham, I/R and NC, respectively