miR-1-3p 表达下调抑制 DDH 软骨细胞的肥大和矿化。
Downregulation of miR-1-3p expression inhibits the hypertrophy and mineralization of chondrocytes in DDH.
机构信息
Department of Orthopedics, The Second Affiliated Hospital of Nanchang University, No. 1 Minde Road, Donghu District, Nanchang, Jiangxi, China.
Department of Pediatrics, The Second Affiliated Hospital of Nanchang University, No. 1 Minde Road, Donghu District, Nanchang, Jiangxi, China.
出版信息
J Orthop Surg Res. 2021 Aug 18;16(1):512. doi: 10.1186/s13018-021-02666-1.
BACKGROUND
Developmental dysplasia of the hip (DDH) is a highly prevalent hip disease among children. However, its pathogenesis remains unclear. MicroRNAs (miRNA) are important regulators of cartilage development. In a previous study, high-throughput miRNA sequencing of tissue samples from an animal model of DDH showed a low level of miR-1-3p in the cartilage of the acetabular roof (ARC), but its role in DDH pathogenesis was not addressed. Therefore, our aim here was to investigate the effects of miR-1-3p in the ARC.
METHODS
The diagnosis of acetabular dysplasia was confirmed with X-ray examination, while imaging and HE staining were conducted to further evaluate the ARC thickness in each animal model. FISH was employed to verify miR-1-3p expression in the ARC and chondrocytes. The miR-1-3p target genes were predicted by a bioinformatics database. A dual-luciferase reporter assay was used to confirm the targeting relationship between miR-1-3p and SOX9. The gene expression of miR-1-3p, SOX9, RUNX2 and collagen type X was evaluated by qPCR analysis. The protein expression of SOX9, RUNX2 and collagen type X was detected by western blot analysis. The levels of SOX9, RUNX2, and collagen type X in the ARC were further assessed via immunohistochemistry analysis. Finally, Alizarin Red S staining was used to observe the mineralized nodules produced by the chondrocytes.
RESULTS
We observed low expression of miR-1-3p in the ARC of animals with DDH. SOX9 is a miR-1-3p target gene. Using miR-1-3p silencing technology in vitro, we demonstrated significantly reduced chondrocyte-generated mineralized nodules compared to those of the control. We also confirmed that with miR-1-3p silencing, SOX9 expression was upregulated, whereas the expression of genes associated with endochondral osteogenesis such as RUNX2 and collagen type X was downregulated. To confirm the involvement of miR-1-3p silencing in abnormal ossification through SOX9, we also performed a rescue experiment in which SOX9 silencing restored the low expression of RUNX2 and collagen type X produced by downregulated miR-1-3p expression. Finally, the elevated SOX9 levels and reduced RUNX2 and collagen type X levels in the ARC of rabbits with DDH were also verified using immunohistochemistry, RT-PCR, and western blots.
CONCLUSION
The relatively low expression of miR-1-3p in the ARC may be the cause of abnormal endochondral ossification in the acetabular roof of animals with DDH.
背景
发育性髋关节发育不良（DDH）是儿童中一种高发的髋关节疾病。然而，其发病机制尚不清楚。微小 RNA（miRNA）是软骨发育的重要调节因子。在之前的一项研究中，对 DDH 动物模型的组织样本进行高通量 miRNA 测序显示，髋臼顶（ARC）软骨中的 miR-1-3p 水平较低，但并未探讨其在 DDH 发病机制中的作用。因此，本研究旨在探讨 miR-1-3p 在 ARC 中的作用。
方法
通过 X 射线检查确诊髋臼发育不良，通过影像学和 HE 染色进一步评估每个动物模型的 ARC 厚度。FISH 用于验证 miR-1-3p 在 ARC 和软骨细胞中的表达。生物信息学数据库预测 miR-1-3p 的靶基因。双荧光素酶报告基因实验用于验证 miR-1-3p 与 SOX9 之间的靶向关系。通过 qPCR 分析评估 miR-1-3p、SOX9、RUNX2 和胶原 X 型的基因表达。通过 Western blot 分析检测 SOX9、RUNX2 和胶原 X 型的蛋白表达。通过免疫组织化学分析进一步评估 ARC 中 SOX9、RUNX2 和胶原 X 型的水平。最后，茜素红 S 染色观察软骨细胞产生的矿化结节。
结果
我们观察到 DDH 动物 ARC 中 miR-1-3p 表达水平较低。SOX9 是 miR-1-3p 的靶基因。在体外使用 miR-1-3p 沉默技术，我们发现与对照组相比，软骨细胞产生的矿化结节明显减少。我们还证实，沉默 miR-1-3p 后，SOX9 表达上调，而与软骨内成骨相关的基因如 RUNX2 和胶原 X 型的表达下调。为了证实 miR-1-3p 沉默通过 SOX9 参与异常成骨，我们还进行了挽救实验，其中沉默 SOX9 恢复了下调 miR-1-3p 表达导致的 RUNX2 和胶原 X 型表达降低。最后，通过免疫组织化学、RT-PCR 和 Western blot 验证了 DDH 兔 ARC 中 SOX9 水平升高和 RUNX2 和胶原 X 型水平降低。
结论
ARc 中 miR-1-3p 的相对低表达可能是 DDH 动物髋臼顶软骨异常软骨内成骨的原因。
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