LncRNA-H19 Modulates Wnt/β-catenin Signaling by Targeting Dkk4 in Hindlimb Unloaded Rat
LncRNA-H19 Modulates Wnt/β-catenin Signaling by Targeting Dkk4 in Hindlimb Unloaded Rat
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LncRNA-H19 在后肢无负荷大鼠中通过靶向 Dkk4 调节 Wnt/β-catenin 信号转导
DOI：
10.1111/os.12321
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发表时间：
2017-08-01
影响因子：
2.1
通讯作者：
Ma, Xin-long
中科院分区：
文献类型：
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作者：
Li, Bing;Liu, Jun;Ma, Xin-long
目的：探讨长链非编码RNA-H19（H19）在废用性骨质疏松（DOP）发病机制中的生物学功能。方法：54只雄性SD大鼠随机分为3组：基线对照组（BC，6）、年龄匹配对照组（AC，24）和后肢减重组（HLU，24）。 BC组大鼠在实验开始时处死，AC组和HLU组大鼠在不同时间（HLU后7、14、21和28天）处死。通过显微CT扫描验证DOP模型，并使用实时定量聚合酶链反应（qRT-PCR）定量成骨基因（OPG、RunX2和OPG）的表达。通过基因测序和生物信息分析来寻找H19靶基因和相关信号通路，并首先在组织样本上进行验证。通过敲低大鼠成骨细胞系（UMR106细胞）中的H19及相关基因进行进一步验证。然后，检查相关信号通路和成骨功能的变化，以证实生物信息学分析的预测。结果：Micro-CT扫描和定量实时聚合酶链反应（qRT-PCR）测试显示HLU期间股骨远端干骺端骨小梁逐渐恶化，成骨基因水平下降，表明DOP模型成功建立。根据RNA测序，1351个mRNA和464个lncRNA因机械卸载而异常表达，其中HLU大鼠中H19减少了2.86倍。预测有1426个mRNA是H19的靶基因，KEGG通路分析表明Wnt信号通路（Wntsignaling）是负责这些靶基因的首要通路。 H19靶向的Wnt相关基因中，有11个在HLU和AC大鼠之间存在差异表达，其中Dkk4在HLU大鼠中比正常对照增加了2.44倍。 DOP 大鼠中观察到的 H19 低表达、Dkk4 过表达和 Wnt 信号传导抑制证实了这些测序和生物信息分析结果。随后的体外细胞测定进一步证明，H19 的敲低导致 Dkk4 上调，并抑制 UMR106 细胞中的 Wnt 信号传导和成骨功能。应用敲低 Dkk4 后，这些效应可以大大逆转。结论：我们的研究结果表明，机械卸载诱导的 H19 低表达可通过促进 Dkk4 表达抑制 Wnt 信号传导，从而导致 DOP 的产生。
Objective: To investigate the biological functions of long noncoding RNA-H19 (H19) in the pathogenesis of disuse osteoporosis (DOP).Methods: Fifty-four male Sprague Dawley (SD) rats were randomly divided into three groups: baseline control (BC, 6), age-matched control (AC, 24), and hindlimb unloading (HLU, 24). The rats in the BC group were sacrificed at the beginning of the experiment, while the AC and HLU rats were sacrificed at different times (7, 14, 21 and 28 days after HLU). The DOP model was verified by micro-CT scan, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to quantify the expression of osteogenic genes (OPG, RunX2 and OPG). Gene sequencing and bioinformatic analysis were performed to find H19 target genes and the associated signaling pathway, which were first verified on tissue samples. Further verification was performed by knocking down the H19 and related gene in rat osteoblast cell line (UMR106 cell). Then, the changes of associated signaling pathway and osteogenic function were examined to confirm the prediction of the bioinformatic analysis.Results: Micro-CT scans and quantitative real-time polymerase chain reaction (qRT-PCR) tests showed progressively deteriorated trabecular bone and decreased level of osteogenic genes in the metaphysis of distal femur during HLU, indicating the successful establishment of a DOP model. According to RNA sequencing, 1351 mRNA and 464 lncRNA were abnormally expressed in response to mechanical unloading, in which the H19 decreased 2.86 fold in HLU rats. There were 1426 mRNA predicted to be the target genes of H19, and KEGG pathway analysis suggested that Wnt signaling pathway (Wnt signaling) was the top pathway responsible for these target genes. In the Wnt-associated genes targeted by H19, 11 were differentially expressed between HLU and AC rats, among which Dkk4 increased 2.44 fold in HLU rats when compared to normal controls. These results of sequencing and bioinformatic analysis were confirmed by the low expression of H19, overexpression of Dkk4 and inhibited Wnt signaling observed in DOP rats. Subsequent in vitro cell assay further demonstrated that knockdown of H19 led to upregulation of Dkk4, and inhibition of Wnt signaling and osteogenic function in UMR106 cell. These effects can be greatly reversed after application of knocking down Dkk4.Conclusion: Our findings demonstrated that low expression of H19, induced by mechanical unloading, leads to development of DOP through inhibition of Wnt signaling by promoting Dkk4 expression.