MiR-335 Improves Functional Recovery in Rats After Spinal Cord Injury and Protects PC12 Cells Against Injury Via the SPI-Bax/Caspase-3 Axis

Study design: Animal and cellular models of spinal cord injury (SCI) were used to explore the role of miR-335 in regulating cell viability and Apoptosis.

Objective: To investigate the role and the target of miR-335 in SCI.

Summary of background: Based on analysis of the GSE19890 data set, miR-335 was identified as a downregulated MicroRNA (miRNAs) following SCI. Thus, this study investigated whether downregulation of miR-335 is important in the pathological process of SCI.

Materials and methods: The GSE19890 data set investigating the expression profiles of miRNAs after SCI was downloaded from the GEO database. GSE45006 and GSE4550 data sets were used to identify differentially expressed genes between normal samples and SCI samples. The targets of rno-miR-335 were predicted using the TargetScan database.An experimental model of SCI was established, and agomir-miR-335 was intrathecally injected into rats with SCI. Functional recovery was evaluated by assessment of Basso-Beattie-Bresnahan scores and spinal cord water content and performing hematoxylin-eosin staining. Apoptosis was estimated by TUNEL staining. The H2O2-treated PC12 cells were used as in vitro models of SCI. Cell viability and Apoptosis were examined by cell counting kit-8 and flow cytometry. Caspase-3 expression was evaluated by immunofluorescence staining. Levels of miRNAs and mRNAs were measured by Reverse Transcriptase quantitative polymerase chain reaction. Western blotting was performed to measure Bcl-2, Bax, cleaved Caspase-3, and specificity protein 1 (SP1) protein levels.

Results: For in vivo studies, miR-335 was downregulated following SCI, and agomir-miR-335 delivery improved functional recovery through suppressing neuronal Apoptosis by inactivating the SP1-Bax/Bcl-2/Caspase-3 signaling. During in vitro analysis, miR-335 protected PC12 cells against H2O2-induced damage by negatively regulating the SP1-Bax/Bcl-2/Caspase-3 signaling axis. Moreover, upregulation of SP1 abolished the Apoptosis suppressive effects of miR-335 upregulation.

Conclusion: MiR-335 ameliorates locomotor impairment in rats with SCI through the suppression of neuronal Apoptosis by inactivating SP1-Bax/Bcl-2/Caspase-3 signaling.