Time-specific microRNA changes during spinal motoneuron degeneration in adult rats following unilateral brachial plexus root avulsion: ipsilateral vs. contralateral changes

Tang et al. BMC Neuroscience 2014, 15:92 http://www.biomedcentral.com/1471-2202/15/92 RESEARCH ARTICLE Open Access Time-specific microRNA changes during spinal motoneuron degeneration in adult rats following unilateral brachial plexus root avulsion: ipsilateral vs. contralateral changes Ying Tang1†, Ze-Min Ling1†, Rao Fu1, Ying-Qin Li1,2, Xiao Cheng1, Fa-Huan Song1, Hao-Xuan Luo1 and Li-Hua Zhou1* Abstract Background: Spinal root avulsion induces multiple pathophysiological events consisting of altered levels of specific genes and proteins related to inflammation, apoptosis, and oxidative stress, which collectively result in the death of the affected motoneurons. Recent studies have demonstrated that the gene changes involved in spinal cord injury can be regulated by microRNAs, which are a class of short non-coding RNA molecules that repress target mRNAs post-transcriptionally. With consideration for the time course of the avulsion-induced gene expression patterns within dying motoneurons, we employed microarray analysis to determine whether and how microRNAs are involved in the changes of gene expression induced by pathophysiological events in spinal cord motoneurons. Results: The expression of a total of 3,361 miRNAs in the spinal cord of adult rats was identified. Unilateral rootavulsion resulted in significant alterations in miRNA expression. In the ipsilateral half compared to the contralateral half of the spinal cord, on the 3rd day after the injury, 55 miRNAs were upregulated, and 24 were downregulated, and on the 14th day after the injury, 36 miRNAs were upregulated, and 23 were downregulated. The upregulation of miR-146b-5p and miR-31a-3p and the downregulation of miR-324-3p and miR-484 were observed. Eleven of the miRNAs, including miR-21-5p, demonstrated a sustained increase; however, only miR-466c-3p presented a sustained decrease 3 and 14 days after the injury. More interestingly, 4 of the miRNAs, including miR-18a, were upregulated on the 3rd day but were downregulated on the 14th day after injury. Some of these miRNAs target inflammatory-response genes in the early stage of injury, and others target neurotransmitter transport genes in the intermediate stages of injury. The altered miRNA expression pattern suggests that the MAPK and calcium signaling pathways are consistently involved in the injury response. Conclusions: This analysis may facilitate the understanding of the time-specific altered expression of a large set of microRNAs in the spinal cord after brachial root avulsion. Keywords: Brachial root avulsion, MicroRNA, Microarray, Inflammatory response, nNOS, c-jun, ATF-3, Calpain 2 * Correspondence: † Equal contributors 1 Department of Anatomy, Zhongshan School of Medicine, Sun Yat-sen University, No. 74 Zhongshan Road 2, Guangzhou 510080, P.R. China Full list of author information is available at the end of the article © 2014 Tang et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tang et al. BMC Neuroscience 2014, 15:92 http://www.biomedcentral.com/1471-2202/15/92 Background Brachial root avulsion is a type of injury that leads to motor function loss as a result of motoneuron degeneration. Previous studies have shown that avulsion-induced motoneuron damage is propagated by a cascade of molecular and cellular events including changes in the expression of genes and the phosphorylation of signaling molecules in cell death-related pathways [1-3]. Based on the microarray analysis of the affected spinal cord after root avulsion in previous studies, the downregulation of genes required for promoting neuronal survival and axonal regeneration and the upregulation of genes involved in apoptosis and DNA damage were observed. Furthermore, our recent studies showed that some avulsion-induced genes, such as neuronal nitric oxide synthase (nNOS), showed changes at the mRNA level that were different from the changes at the protein levels [4,5], whereas other changes, such as those in c-jun, were similar at the protein and mRNA levels [1,3]. However, the upstream and downstream molecular mechanisms of avulsion-induced abnormal gene expression during motoneuron degeneration are still unclear and need to be studied further. MicroRNAs (miRNAs) are small non-coding RNAs that are key determinants of mRNA stability [6]. miRNAs modulate protein expression levels by antagonizing mRNA translation and are powerful regulators of cellular function [7]. Individual miRNAs target and block hundreds of protein-coding genes [8] that regulate many biological processes in neuronal lesions [9]. An increasing number of studies have demonstrated that miRNAs in the spinal cord are altered in a variety of motor neuron degenerative diseases and after spinal cord injury [8,10-12]. There is also emerging evidence that alterations in RNA metabolism in the spinal cord are time-specific and may be critical in the progression of avulsion-induced motoneuron degeneration [3,13]. However, little is known regarding the miRNA expression profile in the spinal cord during avulsion-induced motoneuron degeneration. Therefore, we hypothesize that the use of miRNAs may be an ideal and potent method for determining the underlying mechanism of avulsion-induced motoneuron death. In the present study, we investigated the miRNA expression patterns based on the time course of motoneuron death by using microarray analysis followed by quantitative RTPCR confirmation. We chose two time points, 3 and 14 days after avulsion, when the RNA stability in the spinal cord was lost and when motoneuron death began to occur, respectively, to investigate the miRNA expression patterns. Furthermore, we focused on the changes in the miRNA expression patterns in the ipsilateral vs. contralateral halves of the affected spinal cord. We employed bioinformatics analysis to determine the functional roles of target genes regulated by altered miRNAs, which further suggested the effects of miRNA Page 2 of 14 dysregulation on the key processes of the brachial root avulsion injury. Results MicroRNA expression profiling of the injured rat spinal cord In the present study, 3,361 miRNAs were expressed in the cervical spinal cord of the adult rats. After normalizing the signal intensities for all miRNA expression levels, miR-124-3p, miR-9a-3p, miR-34a-5p, miR-9a-5p, miR125b-5p, miR-let-7c-5p, miR-29a-3p, miR-23b-3p, miR451-5p, and miR-30c-5p were the miRNAs expressed at the highest levels (Figure 1). In the rats with right brachial plexus root avulsion, the miRNA expression patterns of the ips (...truncated)