Department of Cellular & Molecular Medicine and Center for Neuromuscular Disease, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada.
J Neurochem. 2012 Jan;120(2):230-8. doi: 10.1111/j.1471-4159.2011.07583.x. Epub 2011 Dec 6.
Brain-derived neurotrophic factor (BDNF) is required for efficient skeletal-muscle regeneration and perturbing its expression causes abnormalities in the proliferation and differentiation of skeletal muscle cells. In this study, we investigated the mechanism of BDNF suppression that occurs during myogenic differentiation. BDNF is expressed at the mRNA level as two isoforms that differ in the length of their 3'UTRs as a result of alternative cleavage and polyadenylation. Sequence analysis revealed the presence of three miR-206 target sites in the long BDNF 3'UTR (BDNF-L), whereas only one site was found in the short mRNA BDNF 3'UTR (BDNF-S). miR-206 is known to regulate the differentiation of C2C12 myoblasts and its expression is induced during the transition from myoblasts to myotubes. We thus examined whether miR-206-mediated suppression is responsible for the expression pattern of BDNF during myogenic differentiation. BDNF-L was suppressed to a greater extent than BDNF-S during differentiation of C2C12 myoblasts. Transfection of a miR-206 precursor decreased activity of reporters representative of the BDNF-L 3'UTR, but not BDNF-S 3'UTR, and repressed endogenous BDNF mRNA levels. This suppression was found to be dependent on the presence of multiple miR-206 target sites in the BDNF-L 3'UTR. Conversely, suppression of miR-206 levels resulted in de-repression of BDNF 3'UTR reporter activity and increased endogenous BDNF-L mRNA levels. A receptor for BDNF, p75(NTR) , was also suppressed during differentiation and in response to miR-206, but this appeared to not be entirely mediated via a miR-206 target site its 3'UTR. Based on these observations, BDNF represents a novel target through which miR-206 controls the initiation and maintenance of the differentiated state of muscle cells. These results further suggest that miR-206 might play a role in regulating retrograde signaling of BDNF at the neuromuscular junction.
脑源性神经营养因子（BDNF）是有效骨骼肌再生所必需的，其表达的改变会导致骨骼肌细胞增殖和分化异常。在这项研究中，我们研究了肌生成分化过程中 BDNF 抑制的机制。BDNF 在 mRNA 水平上表达为两种异构体，其 3'UTR 的长度因选择性剪接和多聚腺苷酸化而不同。序列分析显示，长 BDNF 3'UTR（BDNF-L）中存在三个 miR-206 靶位点，而短 BDNF mRNA 3'UTR（BDNF-S）中仅存在一个位点。miR-206 已知可调节 C2C12 成肌细胞的分化，其表达在成肌细胞向肌管的转化过程中被诱导。因此，我们检查了 miR-206 介导的抑制是否负责肌生成分化过程中 BDNF 的表达模式。在 C2C12 成肌细胞的分化过程中，BDNF-L 的抑制程度大于 BDNF-S。miR-206 前体的转染降低了代表 BDNF-L 3'UTR 的报告基因的活性，但不降低 BDNF-S 3'UTR 的活性，并抑制内源性 BDNF mRNA 水平。这种抑制被发现依赖于 BDNF-L 3'UTR 中多个 miR-206 靶位点的存在。相反，抑制 miR-206 水平导致 BDNF 3'UTR 报告基因活性的去抑制和内源性 BDNF-L mRNA 水平的增加。BDNF 的一种受体，p75（NTR），也在分化过程中被抑制，并对 miR-206 产生反应，但这似乎不完全是通过其 3'UTR 中的 miR-206 靶位点介导的。基于这些观察结果，BDNF 是一个新的靶标，miR-206 通过它来控制肌肉细胞分化状态的启动和维持。这些结果进一步表明，miR-206 可能在调节神经肌肉接头处 BDNF 的逆行信号传递中发挥作用。
