Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland.
Birmingham Women's and Children's NHS Foundation Trust, Birmingham, United Kingdom.
Endocrinology. 2018 Mar 1;159(3):1469-1478. doi: 10.1210/en.2017-00780.
Growth plate chondrocytes undergo sequential differentiation to form the resting zone, the proliferative zone (PZ), and the hypertrophic zone (HZ). The important role of microRNAs (miRNAs) in the growth plate was previously revealed by cartilage-specific ablation of Dicer, an enzyme essential for biogenesis of many miRNAs. To identify specific miRNAs that regulate differentiation of PZ chondrocytes to HZ chondrocytes, we microdissected individual growth plate zones from juvenile rats and performed miRNA profiling using a solution hybridization method and miRNA sequencing. Thirty-four miRNAs were differentially expressed between the PZ and the HZ, and we hypothesized that some of the miRNAs that are preferentially expressed in the PZ may promote proliferation and inhibit hypertrophic differentiation. Consistent with this hypothesis, transfection of inhibitors for four of these miRNAs (mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p) decreased proliferation in primary epiphyseal chondrocytes. The inhibitors for three of these miRNAs (mir-374-5p, mir-379-5p, and mir-503-5p) also increased expression of multiple genes that are associated with chondrocyte hypertrophic differentiation. We next hypothesized that preferential expression of these miRNAs in the PZ is driven by the parathyroid hormone-related protein (PTHrP) concentration gradient across the growth plate. Consistent with this hypothesis, treatment of primary chondrocytes with a parathyroid hormone (PTH)/PTHrP receptor agonist, PTH1-34, increased expression of mir-374-5p, mir-379-5p, and mir-503-5p. Taken together, our findings suggest that the PTHrP concentration gradient across the growth plate induces differential expression of mir-374-5p, mir-379-5p, and mir-503-5p between the PZ and the HZ. In the PZ, the higher expression levels of these miRNAs promote proliferation and inhibit hypertrophic differentiation. In the HZ, downregulation of these miRNAs inhibits proliferation and promotes hypertrophic differentiation.
生长板软骨细胞经历连续分化，形成静止区、增生区（PZ）和肥大区（HZ）。以前通过软骨特异性消融 Dicer 揭示了 microRNAs（miRNAs）在生长板中的重要作用，Dicer 是许多 miRNAs 生物发生所必需的酶。为了鉴定调节 PZ 软骨细胞向 HZ 软骨细胞分化的特定 miRNAs，我们从小鼠的单个生长板区进行显微解剖，并使用溶液杂交方法和 miRNA 测序进行 miRNA 谱分析。PZ 和 HZ 之间有 34 个 miRNAs 表达差异，我们假设在 PZ 中优先表达的一些 miRNAs 可能促进增殖并抑制肥大分化。与该假说一致，四种 miRNA（mir-369-3p、mir-374-5p、mir-379-5p 和 mir-503-5p）抑制剂的转染降低了原代骺软骨细胞的增殖。这四种 miRNA 中的三种抑制剂（mir-374-5p、mir-379-5p 和 mir-503-5p）也增加了与软骨细胞肥大分化相关的多个基因的表达。我们接下来假设，这些 miRNAs 在 PZ 中的优先表达是由生长板中的甲状旁腺激素相关蛋白（PTHrP）浓度梯度驱动的。与该假说一致，用甲状旁腺激素（PTH）/PTHrP 受体激动剂 PTH1-34 处理原代软骨细胞增加了 mir-374-5p、mir-379-5p 和 mir-503-5p 的表达。总之，我们的研究结果表明，生长板中的 PTHrP 浓度梯度诱导 PZ 和 HZ 之间 mir-374-5p、mir-379-5p 和 mir-503-5p 的差异表达。在 PZ 中，这些 miRNAs 的更高表达水平促进增殖并抑制肥大分化。在 HZ 中，这些 miRNAs 的下调抑制增殖并促进肥大分化。
