Introduction

X-linked hypophosphatemia (XLH), the most common form of inherited rickets, is characterized by renal phosphate (Pi) wasting, abnormal vitamin D metabolism and defective bone mineralization. It is caused by an inactivation mutation in PHEX gene. Other factors, such as microRNA (miRNA), which regulates biological functions by interfering with specific mRNA translation, also contribute in controlling disease pathogenesis. Recent studies indicate the role of miRNAs in osteoblast functions.

Objectives

The main objective of this study is to identify novel miRNAs, regulated by Phex in osteoblast cells, and to determine whether miR-539-3p, a known tumor suppressor, aids in osteogenesis.

Methods

MiRNA expression patterns in control and siPhex treated cells were analyzed by miRNA microarray profiling and quantitative RT-PCR. Effect of miR-539-3p on osteoblast differentiation was achieved by transfection with miR-539-3p and anti-miR in mouse calvarial osteoblasts. MiR-539-3p targets were identified by Luciferase reporter gene assay. LRP-6, Wnt3, Beta catenin, p-beta catenin, GSK3β and Lef-1 protein levels were determined by Western blotting. To investigate the function of miR-539-3p in vivo, animal groups were divided into; sham+PBS (ovary intact), ovariectomized (Ovx)+miC, Ovx+miR and Ovx+anti-miR. Chemically modified oligonucleotide miR and anti-miR were injected at 5ug/animal dose via a subcutaneously injection into mice.

Results

In PHEX silenced mice calvarial osteoblast cells, miR-539-3p was up-regulated as analyzed by miRNA array profiling and qRT-PCR analysis. Over-expression of miR-539-3p inhibited osteoblast differentiation, whereas inhibition of miR-539-3p function promoted expression of osteoblast-specific genes and alkaline phosphatase (ALP) activity. Target prediction analysis tools and experimental validation by luciferase 3â² UTR reporter assay identified LRP-6 as a direct target of miR-539-3p. Over-expression of miR-539-3p led to LRP-6 repression and inhibition of Wnt-Beta catenin signaling. Furthermore, miR-539-3p silencing led to increased bone formation and improved trabecular micro architecture in Ovx mice.

Conclusions

Although miR-539-3p is known to be a tumor repressor, we identified a second complementary function of miR-539-3p where it inhibits LRP-6-mediated osteogenesis. Our findings suggest that pharmacological inhibition of miR-539-3p by anti-miR-539-3p could represent a therapeutic strategy for enhancing bone formation in vivo.

中文翻译：

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MiR-539-3p 通过靶向 LRP-6 抑制体内成骨细胞活性和骨形成

介绍

X 连锁低磷血症 (XLH) 是遗传性佝偻病的最常见形式，其特征是肾磷酸盐 (Pi) 消耗、维生素 D 代谢异常和骨矿化缺陷。它是由 PHEX 基因的失活突变引起的。其他因素，例如通过干扰特定 mRNA 翻译来调节生物学功能的 microRNA (miRNA)，也有助于控制疾病的发病机制。最近的研究表明 miRNA 在成骨细胞功能中的作用。

目标

本研究的主要目的是鉴定成骨细胞中受 Phex 调节的新型 miRNA，并确定已知的肿瘤抑制因子 miR-539-3p 是否有助于成骨。

方法

通过 miRNA 微阵列分析和定量 RT-PCR 分析对照和 siPhex 处理的细胞中的 miRNA 表达模式。miR-539-3p 对成骨细胞分化的影响是通过在小鼠颅骨成骨细胞中转染 miR-539-3p 和 anti-miR 来实现的。MiR-539-3p 靶标通过荧光素酶报告基因测定法进行鉴定。LRP-6、Wnt3、β 连环蛋白、p-β 连环蛋白、GSK3β 和 Lef-1 蛋白水平由蛋白质印迹法测定。为了研究 miR-539-3p 在体内的功能，将动物组分为：假手术+PBS（卵巢完整）、去卵巢（Ovx）+miC、Ovx+miR和Ovx+anti-miR。通过皮下注射将化学修饰的寡核苷酸 miR 和抗 miR 以 5ug/动物剂量注射到小鼠体内。

结果

在 PHEX 沉默的小鼠颅骨成骨细胞中，通过 miRNA 阵列分析和 qRT-PCR 分析分析，miR-539-3p 上调。miR-539-3p 的过表达抑制成骨细胞分化，而 miR-539-3p 功能的抑制促进了成骨细胞特异性基因的表达和碱性磷酸酶 (ALP) 活性。目标预测分析工具和通过荧光素酶 3ℓUTR 报告基因测定的实验验证将 LRP-6 确定为 miR-539-3p 的直接目标。miR-539-3p 的过表达导致 LRP-6 抑制和 Wnt-β 连环蛋白信号传导的抑制。此外，miR-539-3p 沉默导致 Ovx 小鼠骨形成增加和小梁微结构改善。

结论

尽管已知 miR-539-3p 是一种肿瘤抑制因子，但我们确定了 miR-539-3p 的第二个互补功能，它抑制 LRP-6 介导的成骨。我们的研究结果表明，抗 miR-539-3p 对 miR-539-3p 的药理学抑制可能代表了一种增强体内骨形成的治疗策略。