MiR-27a ameliorates inflammatory damage to the blood-spinal cord barrier after spinal cord ischemia: reperfusion injury in rats by downregulating TICAM-2 of the TLR₄ signaling pathway

Spinal cord ischemia reperfusion (IR) injury causes inflammation and subsequently increases blood-spinal cord barrier leakage and Toll-like receptor 4 (TLR₄) pathway activation. MicroRNAs (miRs) effectively regulate numerous target mRNAs during ischemia. However, their roles during IR injury are poorly understood. We investigated miRs involvement, particularly miR-27a, in TLR₄ pathway-mediated inflammatory responses after IR.

We used a genomics approach to examine changed miRs of rats that had undergone 14 minutes of ischemia, followed by 24 or 72 hours of reperfusion. Quantitative RT-PCR was used to identify and confirm the miRs involved in regulating TLR₄ pathway activation. We scanned miR databases for potential miR targets and confirmed these targets by quantitative RT-PCR. The miR mimic and anti-miR oligonucleotides (AMOs) were intrathecally injected at 12-hour intervals beginning three days before the ischemia. The effects of miRs on the TLR₄ pathway and downstream cytokines were analyzed by PCR, western blotting, and ELISA. Double immunofluorescence staining was perfumed to determine the relationship between the targets and TLR₄. Blood-spinal cord barrier (BSCB) permeability was examined using Evans blue (EB) dye.

A microarray analysis revealed that at 24 hours post-injury, three miRs were upregulated (>2.0 fold) and 15 miRs were downregulated (<0.5 fold), and at 72 hours, four miRs were upregulated and 14 were downregulated compared to their levels in sham-operated controls. We focused on miR-27a, which is predicted to contain sequences complementary to the 3'-untranslated region (UTR) of Toll-like receptor adaptor molecule 2 (TICAM-2). Double immunostaining indicated that TLR₄ activation correlated with changes in TICAM-2 expression. Compared to the rats in the IR and negative control groups, intrathecal infusion of the miR-27a mimic attenuated IR-induced TLR₄ activation and inflammatory damage to the BSCB, which was shown as decreased EB extravasation and lower levels of nuclear factor kappa-B (NF-κB) and lnterleukin (IL)-1β at 24 and 72 hours after reperfusion, whereas pretreatment with miR-27a AMO aggravated these injuries.

We present the first evidence that miRs play an important role in spinal cord IR injury. We identified TICAM-2 as a novel target of miR-27a. miR-27a upregulation attenuates IR-induced inflammatory damage to the BSCB by negatively regulating TICAM-2 of the TLR₄ signaling pathway and inhibiting the NF-κB/IL-1β pathway. These results provide new therapeutic targets for IR injury treatment.