Department of Oral Disease Research, National Center for Geriatrics and Gerontology, Obu, Japan.
PLoS One. 2013;8(3):e58796. doi: 10.1371/journal.pone.0058796. Epub 2013 Mar 22.
MicroRNAs (miRNAs) are small RNA molecules of 21-25 nucleotides that regulate cell behavior through inhibition of translation from mRNA to protein, promotion of mRNA degradation and control of gene transcription. In this study, we investigated the miRNA expression signatures of cell cultures undergoing osteoblastic and osteocytic differentiation from mesenchymal stem cells (MSC) using mouse MSC line KUSA-A1 and human MSCs. Ninety types of miRNA were quantified during osteoblastic/osteocytic differentiation in KUSA-A1 cells utilizing miRNA PCR arrays. Coincidently with mRNA induction of the osteoblastic and osteocytic markers, the expression levels of several dozen miRNAs including miR-30 family, let-7 family, miR-21, miR-16, miR-155, miR-322 and Snord85 were changed during the differentiation process. These miRNAs were predicted to recognize osteogenic differentiation-, stemness-, epinegetics-, and cell cycle-related mRNAs, and were thus designated OstemiR. Among those OstemiR, the miR-30 family was classified into miR-30b/c and miR-30a/d/e groups on the basis of expression patterns during osteogenesis as well as mature miRNA structures. In silico prediction and subsequent qRT-PCR in stable miR-30d transfectants clarified that context-dependent targeting of miR-30d on known regulators of bone formation including osteopontin/spp1, lifr, ccn2/ctgf, ccn1/cyr61, runx2, sox9 as well as novel key factors including lin28a, hnrnpa3, hspa5/grp78, eed and pcgf5. In addition, knockdown of human OstemiR miR-541 increased Osteopontin/SPP1 expression and calcification in hMSC osteoblastic differentiation, indicating that miR-541 is a negative regulator of osteoblastic differentiation. These observations indicate stage-specific roles of OstemiR especially miR-541 and the miR-30 family on novel targets in osteogenesis.
微小 RNA（miRNAs）是一种 21-25 个核苷酸的小 RNA 分子，通过抑制从 mRNA 到蛋白质的翻译、促进 mRNA 降解和控制基因转录来调节细胞行为。在这项研究中，我们使用小鼠间充质干细胞（MSC）系 KUSA-A1 和人 MSC 研究了 MSC 向成骨细胞和骨细胞分化过程中的 miRNA 表达特征。利用 miRNA PCR 阵列，在 KUSA-A1 细胞中成骨细胞/骨细胞分化过程中定量了 90 种 miRNA。与成骨细胞和骨细胞标志物的 mRNA 诱导同时发生，包括 miR-30 家族、let-7 家族、miR-21、miR-16、miR-155、miR-322 和 Snord85 在内的数十种 miRNA 的表达水平在分化过程中发生变化。这些 miRNA 被预测能识别成骨分化、干性、表观遗传学和细胞周期相关的 mRNA，因此被命名为 OstemiR。在这些 OstemiR 中，miR-30 家族根据成骨过程中的表达模式和成熟 miRNA 结构被分为 miR-30b/c 和 miR-30a/d/e 组。在稳定转染 miR-30d 的细胞中进行计算机预测和随后的 qRT-PCR 表明，miR-30d 在包括骨形成已知调节剂骨桥蛋白/ SPP1、lifr、ccn2/ctgf、ccn1/cyr61、runx2、sox9 以及包括 lin28a、hnrnpa3、hspa5/grp78、eed 和 pcgf5 在内的新的关键因子上的靶定是依赖于背景的。此外，人 OstemiR miR-541 的敲低增加了 hMSC 成骨分化中的骨桥蛋白/ SPP1 表达和钙化，表明 miR-541 是成骨分化的负调节剂。这些观察结果表明 OstemiR 特别是 miR-541 和 miR-30 家族在成骨过程中对新靶点具有特定阶段的作用。
