Ou Minglin, Zhang Xiaoqing, Dai Yong, Gao Jieying, Zhu Mingsong, Yang Xiangchun, Li Yuchao, Yang Ting, Ding Min
Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Chongqing Medical University, Chongqing, China.
Eur J Hum Genet. 2014 May;22(5):625-32. doi: 10.1038/ejhg.2013.221. Epub 2013 Oct 2.
MicroRNAs aberrantly express in many human diseases including some metabolic bone disorders. They have been found to be associated with osteoclast differentiation and function, which makes them attractive candidates for the therapy of bone. However, the potential clinical application of microRNAs in therapeutics rests heavily upon our in-depth understanding of microRNAs and their targets. To identify potential microRNA-target pairs associated with osteopetrosis, we performed a system approach including deep sequencing, iTRAQ quantitative proteomics, and bioinformatics in the peripheral blood mononuclear cells (PBMCs) taken from patients with osteopetrosis and health donors. Notably, 123 differently expressed microRNAs, 173 differently expressed proteins, and 117 computationally predicted microRNA-target pairs with reciprocally expressed level in PBMCs were found in the two sample groups. Functional annotation identified that the microRNA-target pairs were involved in cell growth, differentiation, cellular signaling network, and the network highlighted the microRNA-target pair of has-miR-320a and ADP ribosylation factor 1 (Arf1) potentially associated with CLCN7 mutations in osteopetrosis. The pair of has-miR-320a and Arf1 was further verified by real-time PCR, western blot, and the interaction between has-miR-320a and its targeted sequence on the Arf1 mRNAs was confirmed by luciferase assay. Collectively, the present study established a new system approach for the investigation of microRNAs, and the microRNA-target pairs, particular has-miR-320a and Arf1, may have important roles in osteopetrosis.
微小RNA在包括一些代谢性骨疾病在内的许多人类疾病中异常表达。它们已被发现与破骨细胞的分化和功能相关，这使其成为有吸引力的骨治疗候选物。然而，微小RNA在治疗学中的潜在临床应用在很大程度上取决于我们对微小RNA及其靶标的深入了解。为了鉴定与骨质石化相关的潜在微小RNA-靶标对，我们采用了一种系统方法，包括对取自骨质石化患者和健康供体的外周血单核细胞（PBMC）进行深度测序、iTRAQ定量蛋白质组学和生物信息学分析。值得注意的是，在两个样本组中发现了123种差异表达的微小RNA、173种差异表达的蛋白质以及117对在PBMC中具有相互表达水平的通过计算预测的微小RNA-靶标对。功能注释表明，这些微小RNA-靶标对参与细胞生长、分化、细胞信号网络，并且该网络突出显示了可能与骨质石化中CLCN7突变相关的has-miR-320a和ADP核糖基化因子1（Arf1）的微小RNA-靶标对。通过实时PCR、蛋白质免疫印迹进一步验证了has-miR-320a和Arf1这一对，并且通过荧光素酶测定证实了has-miR-320a与其在Arf1 mRNA上的靶向序列之间的相互作用。总的来说，本研究建立了一种用于研究微小RNA的新系统方法，并且微小RNA-靶标对，特别是has-miR-320a和Arf1，可能在骨质石化中起重要作用。
