The molecular mechanisms how bone marrow-derived mesenchymal stem cells (BMSCs) differentiate into osteoblast need to be investigated. MicroRNAs (miRNAs) contribute to the osteogenic differentiation of BMSCs. However, the effect of miR-346-5p on osteogenic differentiation of BMSCs is not clear. This study is aimed at elucidating the underlying mechanism by which miR-346-5p regulates osteogenic differentiation of human BMSCs. Results of alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining indicated that upregulation of miR-346-5p suppressed osteogenic differentiation of BMSCs, whereas downregulation of miR-346-5p enhanced this process. The protein levels of the osteoblastic markers Osterix and Runt-related transcription factor 2 (Runx2) were decreased in cells treated with miR-346-5p mimic at day 7 and day 14 after being differentiated. By contrast, downregulation of miR-346-5p elevated the protein levels of Osterix and Runx2. Moreover, a dual-luciferase reporter assay revealed that Transmembrane Protein 9 (TMEM9) was a target of miR-346-5p. In addition, the Western Blot results demonstrated that the TMEM9 protein level was significantly reduced by the miR-346-5p mimic whereas downregulation of miR-346-5p improved the protein level of TMEM9. These results together demonstrated that miR-346-5p served a key role in BMSC osteogenic differentiation of through targeting TMEM9, which may provide a novel target for clinical treatments of bone injury.

中文翻译：

---

MicroRNA-346-5p通过抑制跨膜蛋白9调节骨髓间充质干细胞的分化。

需要研究骨髓来源的间充质干细胞（BMSCs）分化为成骨细胞的分子机制。微小RNA（miRNA）有助于骨髓间充质干细胞的成骨分化。但是，miR-346-5p对BMSCs成骨分化的作用尚不清楚。这项研究旨在阐明miR-346-5p调控人类BMSCs成骨分化的潜在机制。碱性磷酸酶（ALP）和茜素红S（ARS）染色的结果表明，miR-346-5p的上调抑制了BMSCs的成骨分化，而miR-346-5p的下调则增强了该过程。在分化后第7天和第14天，用miR-346-5p模拟物处理的细胞中成骨细胞标志物Osterix和Runt相关转录因子2（Runx2）的蛋白质水平降低。相反，miR-346-5p的下调提高了Osterix和Runx2的蛋白质水平。此外，双重萤光素酶报告基因测定表明跨膜蛋白9（TMEM9）是miR-346-5p的靶标。另外，蛋白质印迹结果表明，miR-346-5p模拟物显着降低了TMEM9蛋白水平，而miR-346-5p的下调则改善了TMEM9的蛋白水平。这些结果共同证明，miR-346-5p在通过靶向TMEM9的BMSC成骨分化中起关键作用，这可能为骨损伤的临床治疗提供新的靶点。Western Blot结果表明，miR-346-5p模拟物显着降低了TMEM9的蛋白水平，而miR-346-5p的下调则改善了TMEM9的蛋白水平。这些结果共同证明，miR-346-5p在通过靶向TMEM9的BMSC成骨分化中起关键作用，这可能为骨损伤的临床治疗提供新的靶点。Western Blot结果表明，miR-346-5p模拟物显着降低了TMEM9的蛋白水平，而miR-346-5p的下调则改善了TMEM9的蛋白水平。这些结果共同证明，miR-346-5p在通过靶向TMEM9的BMSC成骨分化中起关键作用，这可能为骨损伤的临床治疗提供新的靶点。