As multipotent progenitor cells, mesenchymal stem cells (MSCs) can renew themselves and give rise to multiple lineages including osteoblastic, chondrogenic and adipogenic lineages. It's previously shown that BMP9 is the most potent BMP and induces osteogenic and adipogenic differentiation of MSCs. However, the molecular mechanism through which BMP9 regulates MSC differentiation remains poorly understood. Emerging evidence indicates that noncoding RNAs, especially microRNAs, may play important roles in regulating MSC differentiation and bone formation. As highly conserved RNA binding proteins, Argonaute (AGO) proteins are essential components of the multi-protein RNA-induced silencing complexes (RISCs), which are critical for small RNA biogenesis. Here, we investigate possible roles of AGO proteins in BMP9-induced lineage-specific differentiation of MSCs. We first found that BMP9 up-regulated the expression of Ago1, Ago2 and Ago3 in MSCs. By engineering multiplex siRNA vectors that express multiple siRNAs targeting individual Ago genes or all four Ago genes, we found that silencing individual Ago expression led to a decrease in BMP9-induced early osteogenic marker alkaline phosphatase (ALP) activity in MSCs. Furthermore, we demonstrated that simultaneously silencing all four Ago genes significantly diminished BMP9-induced osteogenic and adipogenic differentiation of MSCs and matrix mineralization, and ectopic bone formation. Collectively, our findings strongly indicate that AGO proteins and associated small RNA biogenesis pathway play an essential role in mediating BMP9-induced osteogenic differentiation of MSCs.

作为多能祖细胞，间充质干细胞 (MSCs) 可以自我更新并产生多个谱系，包括成骨细胞、软骨细胞和脂肪细胞谱系。先前的研究表明，BMP9 是最有效的 BMP，可诱导 MSC 的成骨和成脂分化。然而，BMP9 调节 MSC 分化的分子机制仍然知之甚少。新出现的证据表明，非编码 RNA，尤其是 microRNA，可能在调节 MSC 分化和骨形成中起重要作用。作为高度保守的 RNA 结合蛋白，Argonaute (AGO) 蛋白是多蛋白 RNA 诱导的沉默复合物 (RISC) 的重要组成部分，对小 RNA 的生物发生至关重要。这里，我们研究了 AGO 蛋白在 BMP9 诱导的 MSCs 谱系特异性分化中的可能作用。我们首先发现 BMP9 上调了MSCs 中的Ago1、Ago2和Ago3。通过设计表达针对单个Ago基因或所有四个Ago基因的多个 siRNA 的多重 siRNA 载体，我们发现沉默单个Ago表达会导致 MSCs 中 BMP9 诱导的早期成骨标志物碱性磷酸酶 (ALP) 活性降低。此外，我们证明同时沉默所有四个Ago基因显着减少了 BMP9 诱导的 MSCs 成骨和成脂分化以及基质矿化，和异位骨形成。总的来说，我们的研究结果强烈表明 AGO 蛋白和相关的小 RNA 生物发生途径在介导 BMP9 诱导的 MSCs 成骨分化中起重要作用。