Silencing of Long Non-Coding RNA NONHSAT009968 Ameliorates the Staphylococcal Protein A-Inhibited Osteogenic Differentiation in Human Bone Mesenchymal Stem Cells
Silencing of Long Non-Coding RNA NONHSAT009968 Ameliorates the Staphylococcal Protein A-Inhibited Osteogenic Differentiation in Human Bone Mesenchymal Stem Cells
复制标题
长非编码 RNA NONHSAT009968 的沉默可改善葡萄球菌蛋白 A 抑制的人骨间充质干细胞的成骨分化
DOI：
10.1159/000447839
复制
发表时间：
2016-01-01
影响因子：
--
通讯作者：
Xu, Yongqing
中科院分区：
文献类型：
--
作者：
Cui, Yi;Lu, Sheng;Xu, Yongqing
背景/目的：骨髓炎定义为骨骼和骨髓的炎症。炎症微环境减弱干细胞的成骨分化能力，抑制成骨细胞介导的骨形成，导致净骨丢失。然而，在骨髓炎的炎症微环境中，长链非编码RNA（lncRNA）对干细胞成骨分化的整个表达谱、功能和副作用尚不清楚。研究方法：本研究以不同浓度的葡萄球菌蛋白A（Staphylococcal protein A，SpA）刺激人骨髓间充质干细胞（human bone mesenchymal stem cells，hBMSCs），诱导体外炎症微环境，部分复制骨髓炎的炎症微环境，并通过ELISA检测炎症细胞因子证实了这一点。用lncRNA微阵列分析了在SpA引发的炎症微环境中hBMSCs成骨分化过程中lncRNA和mRNA的完整表达谱。通过定量逆转录PCR分析（qRT-PCR）验证LncRNA表达水平。用表达NONHSAT 009968-shRNA的慢病毒感染hB-MSCs，使NONHSAT 009968的表达沉默。Western blot检测Runx 2、OCN、OPN、COL 1A 1的表达及碱性磷酸酶（ALP）活性。茜素红染色及碱性磷酸酶（ALP）活性检测。结果：ELISA结果显示，SpA处理诱导了炎症细胞因子IL-1A、IL-6和TNFA的分泌。茜素红染色和碱性磷酸酶检测结果显示，SpA处理抑制了hBMSCs的成骨分化。与对照组相比，共发现2033个lncRNA在SpA处理的hBMSCs中异常表达。在这些lncRNA中，641个下调，1392个上调。基于qRT-PCR的结果，选择lncRNA NONHSAT 009968用于进一步研究。茜素红染色、碱性磷酸酶活性检测以及Runx 2、OCN、OPN、COL 1A 1和碱性磷酸酶的western blot检测结果表明，NONHSAT 009968沉默改善了SpA抑制的hBMSCs成骨分化。结论：我们的研究为将来分析lncRNA在SpA引发的炎症环境中成骨细胞分化中的作用提供了基础，lncRNA NONHSAT 009968可能是促进成骨细胞形成的新靶点。
Background/Aims: Osteomyelitis is defined as an inflammation of the bones and bone marrow. The inflammatory microenvironment attenuates the osteogenic differentiation capacity of stem cells and inhibits osteoblast-mediated bone formation, leading to net bone loss. However, the whole expression profile, function and side effect of long non-coding RNAs (lncRNAs) on osteogenic differentiation of stem cells in an inflammatory microenvironment of osteomyelitis are not known. Methods: In the present study, human bone mesenchymal stem cells (hBMSCs) were treated with different concentrations of Staphylococcal protein A (SpA) to trigger an inflammatory microenvironment in vitro to partly duplicate the inflammatory microenvironment of osteomyelitis, which was confirmed using ELISA for detecting the inflammatory cytokines. The complete expression profiles of lncRNAs and mRNA during osteogenic differentiation of hBMSCs in an inflammatory microenvironment triggered by SpA were analyzed using a lncRNA microarray. LncRNA expression levels were verified by quantitative reverse transcription PCR analysis (qRT-PCR). The expression of NONHSAT009968 in hB-MSCs was silenced by infection with lentivirus expressing NONHSAT009968-shRNA. The expression of Runx2, OCN, OPN, COL1A1, and alkaline phosphatase (ALP) activity was detected by western blot. Alizarin red staining and ALP activity detection were carried out. Results: The results of ELISA showed that SpA treatment induced secretion of inflammatory cytokines IL-1A, IL-6, and TNFA. The results of alizarin red staining and ALP detection showed that SpA treatment suppressed the osteogenic differentiation of hBMSCs. A total of 2033 lncRNAs were found with aberrant expression in SpA-treated hBMSCs compared to controls. Among these lncRNAs, 641 were down-regulated and 1392 were up-regulated. Based on the results of qRT-PCR, lncRNA NONHSAT009968 was chosen for further investigation. The results of alizarin red staining, ALP activity detection, and western blot detection of Runx2, OCN, OPN, COL1A1, and ALP indicated that NONHSAT009968 silencing ameliorates SpA-inhibited osteogenic differentiation in hBMSCs. Conclusion: Our present study provides a basis for future analyses of the role of lncRNAs in osteoblastic differentiation in an inflammatory environment triggered by SpA, and lncRNA NONHSAT009968 might be a new target for promoting osteoblast formation.