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

Ning Bao; Bo Fang; Huangwei Lv; Yanhua Jiang; Fengshou Chen; Zhilin Wang; Hong Ma

doi:10.1007/s10571-018-0597-2

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

Cell Mol Neurobiol. 2018 Aug;38(6):1293-1303. doi: 10.1007/s10571-018-0597-2. Epub 2018 Jun 13.

Authors

Ning Bao^{1

2}, Bo Fang¹, Huangwei Lv¹, Yanhua Jiang¹, Fengshou Chen¹, Zhilin Wang¹, Hong Ma³

Affiliations

¹ Department of Anesthesiology, First Affiliated Hospital, China Medical University, No. 155, North Nanjing Street, Heping District, Shenyang, Liaoning, People's Republic of China.
² Shenyang Women's and Children's Hospital, No. 87 Danan Street, Shenhe Dinstrict, Shenyang, Liaoning, People's Republic of China.
³ Department of Anesthesiology, First Affiliated Hospital, China Medical University, No. 155, North Nanjing Street, Heping District, Shenyang, Liaoning, People's Republic of China. mahong5466@yahoo.com.

PMID: 29948551
DOI: 10.1007/s10571-018-0597-2

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.

MeSH terms

Animals
Apoptosis / drug effects
Disease Models, Animal
Down-Regulation*
Endothelin-Converting Enzymes / metabolism*
MicroRNAs / genetics*
Neurons / metabolism
Rats, Sprague-Dawley
Reperfusion Injury / genetics*
Reperfusion Injury / metabolism
Spinal Cord / metabolism
Spinal Cord Injuries / metabolism
Transcriptional Activation / genetics
Up-Regulation*

Substances

MIRN199 microRNA, rat
MicroRNAs
Endothelin-Converting Enzymes