Molecular Medicine Research Group, Robarts Research Institute, Western University, London, Ontario, Canada.
Molecular Medicine Research Group, Robarts Research Institute, Western University, London, Ontario, Canada ; Department of Clinical Neurological Sciences, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada.
PLoS One. 2014 Jan 15;9(1):e85653. doi: 10.1371/journal.pone.0085653. eCollection 2014.
Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by progressive motor neuron degeneration and neurofilament aggregate formation. Spinal motor neurons in ALS also show a selective suppression in the levels of low molecular weight neurofilament (NEFL) mRNA. We have been interested in investigating the role of microRNAs (miRNAs) in NEFL transcript stability. MiRNAs are small, 20-25 nucleotide, non-coding RNAs that act as post-transcriptional gene regulators by targeting the 3' untranslated region (3'UTR) of mRNA resulting in mRNA decay or translational silencing. In this study, we characterized putative novel miRNAs from a small RNA library derived from control and sporadic ALS (sALS) spinal cords. We detected 80 putative novel miRNAs, 24 of which have miRNA response elements (MREs) within the NEFL mRNA 3'UTR. From this group, we determined by real-time PCR that 10 miRNAs were differentially expressed in sALS compared to controls. Functional analysis by reporter gene assay and relative quantitative RT-PCR showed that two novel miRNAs, miR-b1336 and miR-b2403, were downregulated in ALS spinal cord and that both stabilize NEFL mRNA. We confirmed the direct effect of these latter miRNAs using anit-miR-b1336 and anti-miR-b2403. These results demonstrate that the expression of two miRNAs (miRNAs miR-b1336 and miR-b2403) whose effect is to stabilize NEFL mRNA are down regulated in ALS, the net effect of which is predicted to contribute directly to the loss of NEFL steady state mRNA which is pathognomic of spinal motor neurons in ALS.
肌萎缩侧索硬化症（ALS）是一种致命疾病，其特征是运动神经元进行性退化和神经丝聚集形成。ALS 中的脊髓运动神经元还表现出低分子量神经丝（NEFL）mRNA 水平的选择性抑制。我们一直对研究 microRNAs（miRNAs）在 NEFL 转录本稳定性中的作用感兴趣。miRNAs 是 20-25 个核苷酸的小非编码 RNA，通过靶向 mRNA 的 3'非翻译区（3'UTR）发挥作为转录后基因调节剂的作用，导致 mRNA 降解或翻译沉默。在这项研究中，我们从对照和散发性 ALS（sALS）脊髓的小 RNA 文库中鉴定出了推定的新型 miRNA。我们检测到 80 个推定的新型 miRNA，其中 24 个在 NEFL mRNA 3'UTR 内具有 miRNA 反应元件（MRE）。在这一组中，我们通过实时 PCR 确定与对照组相比，sALS 中有 10 个 miRNA 表达不同。通过报告基因测定和相对定量 RT-PCR 的功能分析表明，两种新型 miRNA，miR-b1336 和 miR-b2403，在 ALS 脊髓中下调，并且两者都稳定 NEFL mRNA。我们使用 anti-miR-b1336 和 anti-miR-b2403 证实了这些 miRNA 的直接作用。这些结果表明，两种 miRNA（miRNA miR-b1336 和 miR-b2403）的表达下调，其作用是稳定 NEFL mRNA，这直接导致 ALS 中脊髓运动神经元的 NEFL 稳态 mRNA 丢失，这是 ALS 中脊髓运动神经元的特征性表现。
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