Sun Junkui, Wang Yisheng, Li Yuebai, Zhao Guoqiang
Department of Orthopaedic Surgery, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China.
J Transl Med. 2014 Jun 14;12:168. doi: 10.1186/1479-5876-12-168.
Human bone marrow mesenchymal stem cells (hBMSCs) are multipotent cells that can differentiate into a variety of cell types. Elevated expression of peroxisome proliferator-activated receptor-γ (PPARγ) promotes the adipogenic differentiation of hBMSCs, and reduces their osteogenic differentiation. MicroRNAs (miRNAs) have been shown to play important roles in the regulation of hBMSCs differentiation. Because bioinformatic analysis has indicated that PPARγ is a candidate target of miR-548d-5p, the aim of this study was to assess the impact of miR-548d-5p on the dexamethasone-induced adipogenic differentiation of hBMSCs.
A quantitative RT-PCR (qRT-PCR) assay was used to compare miR-548d-5p expression levels in dexamethasone-induced hBMSCs and uninduced control cells. Oil red O staining, cellular triglyceride (TG) content, and the mRNA and protein levels of PPARγ and CCAAT/enhancer binding protein α (C/EBPα) were used to evaluate the adipogenic differentiation of hBMSCs. Alkaline phosphatase (ALP) activity and levels of osteocalcin (OCN) and Runx2 were used to evaluate the osteogenic potential of hBMSCs.
Compared with untreated cells, miR-548d-5p expression levels were downregulated during dexamethasone-induced adipogenic differentiation of hBMSCs. In contrast to the profuse Oil Red O staining in the cytoplasm of dexamethasone + scrambled miRNA-treated cells, there was limited staining in the cytoplasm of dexamethasone + miR-548d-5p-treated cells, indicating the absence of adipocytes. Moreover, compared with scrambled miRNA-treated cells, treatment with miR-548d-5p suppressed cellular levels of PPARγ and C/EBPα mRNA and protein, and cell TG content (P < 0.05). In contrast, compared with scrambled miRNA-treated cells, cellular levels of OCN and Runx2 mRNA and protein, as well as ALP activity, were significantly higher in miR-548d-5p-treated cells (P < 0.05). Western blot and luciferase reporter assays confirmed that miR-548d-5p directly targeted the 3'-untranslated region of PPARγ.
miR-548d-5p is downregulated during dexamethasone-induced adipogenic differentiation of hBMSCs. By directly targeting and downregulating PPARγ, miR-548d-5p suppresses the dexamethasone-induced adipogenic differentiation of hBMSCs and enhances their osteogenic potential. Our findings suggest that miR-548d-5p has potential in the treatment of corticosteroid-induced osteonecrosis of the femoral head.
人骨髓间充质干细胞（hBMSCs）是多能细胞，可分化为多种细胞类型。过氧化物酶体增殖物激活受体γ（PPARγ）表达升高促进hBMSCs的成脂分化，并降低其成骨分化。微小RNA（miRNAs）已被证明在hBMSCs分化的调节中起重要作用。由于生物信息学分析表明PPARγ是miR-548d-5p的候选靶标，本研究的目的是评估miR-548d-5p对地塞米松诱导的hBMSCs成脂分化的影响。
采用定量逆转录聚合酶链反应（qRT-PCR）分析比较地塞米松诱导的hBMSCs和未诱导的对照细胞中miR-548d-5p的表达水平。用油红O染色、细胞甘油三酯（TG）含量以及PPARγ和CCAAT/增强子结合蛋白α（C/EBPα）的mRNA和蛋白水平评估hBMSCs的成脂分化。用碱性磷酸酶（ALP）活性以及骨钙素（OCN）和Runx2的水平评估hBMSCs的成骨潜能。
与未处理的细胞相比，在hBMSCs地塞米松诱导的成脂分化过程中miR-548d-5p表达水平下调。与地塞米松+乱序miRNA处理细胞的细胞质中大量油红O染色形成对比，地塞米松+miR-548d-5p处理细胞的细胞质中染色有限，表明不存在脂肪细胞。此外，与乱序miRNA处理的细胞相比，用miR-548d-5p处理可抑制细胞中PPARγ和C/EBPα mRNA及蛋白水平以及细胞TG含量（P<0.05）。相反，与乱序miRNA处理的细胞相比，miR-548d-5p处理的细胞中OCN和Runx2 mRNA及蛋白水平以及ALP活性显著更高（P<0.05）。蛋白质印迹和荧光素酶报告基因检测证实miR-548d-5p直接靶向PPARγ的3'非翻译区。
在hBMSCs地塞米松诱导的成脂分化过程中miR-548d-5p下调。通过直接靶向并下调PPARγ，miR-548d-5p抑制地塞米松诱导的hBMSCs成脂分化并增强其成骨潜能。我们的研究结果表明miR-548d-5p在治疗糖皮质激素诱导的股骨头坏死方面具有潜力。
