Genomic Medicine, Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark.
Connect Tissue Res. 2013;54(3):197-203. doi: 10.3109/03008207.2013.781594. Epub 2013 Apr 15.
microRNAs (miRNAs) are short ∼22 nucleotide RNA sequences that regulate messengerRNA translation. miRNAs have shown to play a role in synthesis of inflammatory mediators. Since inflammation play a role in intervertebral disk (IVD) degeneration, the objective was to isolate miRNA from human lumbar intervertebral disks and subsequently characterize the difference in miRNA expression between the annulus fibrosus (AF) and nucleus pulposus (NP).
Fourteen patients undergoing anterior interbody fusion for degenerative disk disease of the lumbar spine were included. During surgery biopsies from the intervertebral disks were obtained and immediately placed in RNAlater. The RNAlater was decanted and the samples frozen at -80˚C until RNA extraction. This was performed using the Trizol method. Global miRNA expression analysis was performed using the Affymetrix GeneChip® miRNA array.
We developed a method allowing the extraction of miRNA from human intervertebral disks usually yielding 1-4 µg of total RNA pr. 100 mg of disk. Twenty-seven miRNAs had a higher expression in the AF and 10 had the highest expression in the NP. Among the top 15 signaling pathways most likely to be controlled by these miRNAs were the transforming growth factor β (TGFβ), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF) epidermal growth factor (EGF), and actin cytoskeletal pathway.
We have demonstrated the presence of miRNA in the human IVD. The miRNA expression differs from muscle tissue and there are differences between the miRNA expressed in the NP and AF. The miRNAs identified control signaling pathways important for maintenance of the IVD. Future studies may determine the importance of miRNA in the development of IVD disease.
microRNAs（miRNAs）是一种约 22 个核苷酸的短 RNA 序列，可调节信使 RNA 翻译。miRNAs 已被证明在炎症介质的合成中发挥作用。由于炎症在椎间盘（IVD）退变中起作用，因此本研究的目的是从人腰椎间盘分离 miRNA，随后表征纤维环（AF）和髓核（NP）之间 miRNA 表达的差异。
纳入 14 例因腰椎退行性疾病而行前路椎体间融合术的患者。术中从椎间盘取活检，并立即置于 RNAlater 中。倒出 RNAlater，将样本在-80°C 下冷冻直至提取 RNA。使用 Trizol 法进行。使用 Affymetrix GeneChip® miRNA 阵列进行全 miRNA 表达分析。
我们开发了一种从人椎间盘提取 miRNA 的方法，通常从 100mg 椎间盘提取 1-4µg 总 RNA。27 种 miRNA 在 AF 中表达较高，10 种 miRNA 在 NP 中表达最高。在最有可能受这些 miRNA 调控的前 15 个信号通路中，包括转化生长因子β（TGFβ）、血小板衍生生长因子（PDGF）、胰岛素样生长因子（IGF）、表皮生长因子（EGF）和肌动蛋白细胞骨架途径。
我们已经证明了 miRNA 存在于人 IVD 中。miRNA 的表达与肌肉组织不同，并且 NP 和 AF 之间表达的 miRNA 也存在差异。鉴定出的 miRNA 控制着维持 IVD 的信号通路。未来的研究可能会确定 miRNA 在 IVD 疾病发展中的重要性。
