MicroRNA-involved Mechanism In Spinal Cord Ischemia-reperfusion Injury

BackgroundThe ischemia-reperfusion (I/R) injury is a common pathophysiological process in various clinical setting. Spinal cord I/R can play important roles in both spinal cord trauma and iatrogenic injuries, such as surgical complications in aortic aneurysm surgery or spine surgery. The consequence of spinal cord I/R injury can be severe and persistent, and outcome of the existing treatments for spinal cord I/R injury has been unsatisfactory. So it is of great importance to search new way to prevent or treat spinal cord I/R injury. However, lack of studies on mechanism is an obstacle to explore new approach. Thus, further study on the mechanism of spinal cord ischemia-reperfusion injury helps to develop new research ideas.The discovery of miRNAs, which are endogenous, non-coding20-24nt RNA molecules, has enhanced our understanding of gene expression at the post-transcriptional level. A number of miRNAs were found in the mammalian CNS, including the brain and spinal cord, where they are involved in the regulation of nervous diseases and in neurotraumatic pathologies. The role of miRNAs in spinal cord I/R is unknown. Furthermore, the beneficial effects of statins on ischemia-reperfusion injury have been reported in various organs, including the spine, brain, heart, lungs, liver, kidneys and intestines. However, the potential mechanism for statins to promote neurologic recovery after spinal cord ischemia-reperfusion injury has been studied in only a limited number of reports and is still unclear.ObjectiveTo explore the regulatory pathway involving microRNA with statins precondition in spinal cord ischemia-reperfusion(I/R) injury; then further determine the exact mechanism of miRNA to modulate I/R in PC12cells.Methods1. Altered microRNA expression in the ischemic-reperfusion spinal cord with atorvastatin therapySpinal cord ischemia was induced in male SD rats by occluding the descending thoracic aorta. Experimental groups (n=6per group) were as follows:sham operation, control (receiving only normal saline), and atorvastatin (10mg/kg/day for2weeks before occlusion). Neurological function was assessed at6,12,24, and48h (hour) after reperfusion. After48h reperfusion, spinal cords were harvested for hematoxylin-eosin (HE) and2,3,5,-triphenyltetrazolium chloride (TTC) staining. miRNA profile was also determined from isolated RNA using miRNA microarrays, followed by validation with quantitative RT-PCR (qRT-PCR). 2. The changes of miR-146a in PC12cells after I/R injuryCellular ischemia-reperfusion model of PC12cells was established using the way of OGD and then reoxygenation; pO2of cell culture was detected using Blood Gas Analyzer; the mRNA expressions of interleukin-1receptor-associated kinase1(IRAK1) and tumor necrosis factor alpha (TNF-a) in both control and ischemia-reperfusion group were measured using real-time PCR; the cell proliferation assay at the time of0、24、48and72h after ischemia-reperfusion was detected using MTT; at last, we explored the miR-146a and miR-199a-3p expression at the time of6,12or24hours after ischemia-reperfusion injury.3. The mechanism of miR-146a to modulate I/R injury in PC12cellsCellular ischemia-reperfusion model of PC12cells was established using the way of OGD and then reoxygenation; the expression levels of miR-146a were inhibited or over-expressed by transfecting miR-146a inhibitor or mimics; the cell proliferation assay at the time of0、24、48and72h after ischemia-reperfusion was detected using MTT; the bioinformatics software and database were applied to predict the target genes of miR-146a, the vector contained the target gene3’-UTR portion cloned into a luciferase reporter construct, which was co-transfected into HEK-293T with miR-146a mimics; the mRNA expressions of interleukin-1receptor-associated kinase1(IRAK1) and SIKE1in both control and ischemia-reperfusion group were measured using real-time PCR; at last, the expression of IκB-α and phospho-IκB-α were detected by western blot.Results1. Altered microRNA expression in the ischemic-reperfusion spinal cord with atorvastatin therapySpinal cord ischemia thus induced was marked by neurological dysfunction, spinal infarction, and neural cell necrosis in animals. Atorvastatin pretreatment reduced rat spinal cord ischemia-reperfusion injury, and improved neurological function recovery. We also uncovered a previously unknown alteration in temporal expression of a large set of miRNAs following a spinal cord ischemia-reperfusion injury. The target genes for the differentially expressed miRNA include genes encoding components that are involved in the inflammation, apoptosis, and neural damage that are known to play important roles in the pathogenesis of I/R injury. Atorvastatin pretreatment can restore part of the up or down regulations in IRI, such as miR-323and miR-199a.2. The changes of miR-146a in PC12cells after I/R injury2.1pO2of the cell culture before hypoxia was87.36±2.58mmHg, which decreased by56.9% after hypoxia for24h.2.2We observed that the mRNA expression of IRAK1and TNF-α were significantly increased24h after ischemia-reperfusion injury in PC12cells. 2.3The cell proliferation detected by MTT was observed in control group and3different time-points. We found that Ischemia-reperfusion injury inhibited cell proliferation significantly, when compared with the control group, the cell proliferation decreased by17.8%、15.5%and12.9%in24,48and72time-point respectively.2.4Ischemia-reperfusion injury had no effect on the expression of miR-199a-3p; however, the expression of miR-146a elevated significantly, and correlated with the time of hypoxia. In24h-treated group, the miR-146a expression was9times of the control group.3. The mechanism of miR-146a to modulate I/R injury in PC12cells3.1The miR-146a expression decreased significantly at48and72h after miR-146a inhibitor transfection(all P<0.001), however, the miR-146a expression increased significantly at48and72h after miR-146a mimics transfection(all P<0.001).3.2When compared with negative control, the IRAKI mRNA expression was increased significantly after miR-146a inhibitor transfection, which was decreased after mimics transfection (all P<0.05).3.3The expression of phospho-IκB-α decreased by53%in the cells transfected with miR-146a mimics; however, phospho-IκB-α expression increased3.12±0.25fold after miR-146a inhibitor transfection.3.4When compared with negative control, the proliferation of cells transfected with miR-146a inhibitor was inhibited significantly, especially at48and72h after transfection; the cell proliferation increased at24h after miR-146a over-expression by mimics transfection.3.5Dual luciferase reporter gene assay system showed that miR-146a significantly inhibited the reporter gene activity containing SIKE13’-UTR, however, the reporter gene activity containing SIKE13’-UTR mutation didn’t changed.3.6When compared with negative control, the SIKE1mRNA expression was increased significantly after miR-146a inhibitor transfection, which was decreased after mimics transfection (all P<0.05).Conclusions1. Altered expression of miRNAs may contribute to the mechanism of neuroprotection of statins in spinal cord I/R injury and are potential targets for therapeutic interventions following I/R injury.2. In the ischemia-reperfusion injury model of PC12cells, we found that elevated miR-146a expression was correlated with the time of hypoxia, and showed a peak at24hours, which suggested that miR-146a may participate in ischemia-reperfusion injury in PC12cells.3. It has been proven that miR-146a can modulate ischemia-reperfusion induced inflammation in PC12cells through inhibiting IRAK1and NF-κB activity which in turn change TLR signaling pathway.4. We also confirmed that miR-146a modulate proliferation by targeting SIKE1mRNA and inhibiting SIKE1expression in PC12cells.