Objective

To discuss the effect of miR-183 on osteoblast differentiation in the osteoporosis progression via targeting Smad4.

Methods

Osteoporosis models were constructed on ovariectomized (OVX) mice to determine the expression of miR-183 and Smad4. Then, MC3T3-E1 cells and primary osteoblasts were divided into Mock, miR-control, miR-183 mimic, miR-183 inhibitor, siSmad4 and miR-183 inhibitor + siSmad4 groups. Alkaline phosphatase (ALP) staining were performed to determine ALP activity, alizarin red staining to evaluate the calcium deposit, while qRT-PCR and Western blotting were used to determine the expression of related molecules. Besides, MC3T3-E1 cells transfected with miR-control or miR-183 mimic were cultured with or without TGF-β1 to verify whether miR-183 regulates the TGF-β signaling pathway.

Results

MiR-183 was up-regulated with decreased Smad4 in the femur of OVX mice, and dual luciferase reporter gene assay showed that Smad4 was a target of miR-183. As compared to Mock group, MC3T3-E1 cells and primary osteoblasts in the miR-183 mimic group and siSmad4 group had significant reductions of OCN, OPN, Runx2 and Osx, as well as decreased ALP activity and calcium deposit. Contrarily, miR-183 and Smad4 were up-regulated and down-regulated respectively. However, cells in the miR-183 inhibitor group manifested the opposite changes. Besides, osteoblast differentiation in the miR-183 inhibitor + siSmad4 group was weakened evidently when compared to miR-183 inhibitor group. Pathway analysis indicated that miR-183 regulated osteogenic differentiation via TGF-β signaling pathway.

Conclusion

MiR-183 was up-regulated in osteoporosis, and miR-183 overexpression can inhibit osteoblast differentiation by targetedly down-regulating TGF-β pathway member Smad4 to trigger osteoporosis.

中文翻译：

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MiR-183 通过靶向 Smad4 调控成骨细胞在骨质疏松发展中的分化

客观的

探讨miR-183通过靶向Smad4对骨质疏松进展中成骨细胞分化的影响。

方法

在去卵巢 (OVX) 小鼠上构建骨质疏松症模型以确定 miR-183 和 Smad4 的表达。然后，将MC3T3-E1细胞和原代成骨细胞分为Mock、miR-control、miR-183 mimic、miR-183 inhibitor、siSmad4和miR-183 inhibitor + siSmad4组。进行碱性磷酸酶（ALP）染色以确定ALP活性，茜素红染色评估钙沉积，而qRT-PCR和Western印迹用于确定相关分子的表达。此外，在有或没有TGF-β1的情况下培养转染了miR-control或miR-183模拟物的MC3T3-E1细胞，以验证miR-183是否调节TGF-β信号通路。

结果

OVX 小鼠股骨中 MiR-183 上调，Smad4 降低，双荧光素酶报告基因检测显示Smad4是 miR-183 的靶标。与Mock组相比，miR-183模拟组和siSmad4组的MC3T3-E1细胞和原代成骨细胞OCN、OPN、Runx2和Osx显着降低，ALP活性和钙沉积降低。相反，miR-183和Smad4分别上调和下调。然而，miR-183抑制剂组的细胞表现出相反的变化。此外，与miR-183抑制剂组相比，miR-183抑制剂+siSmad4组的成骨细胞分化明显减弱。通路分析表明，miR-183通过TGF-β 信号通路调节成骨分化。

结论

MiR-183 在骨质疏松症中上调，miR-183 过表达可通过靶向下调 TGF-β 通路成员 Smad4 来抑制成骨细胞分化，从而引发骨质疏松症。