Differential expression profiles of long noncoding RNAs and mRNAs in human bone marrow mesenchymal stem cells after exposure to a high dosage of dexamethasone.
Differential expression profiles of long noncoding RNAs and mRNAs in human bone marrow mesenchymal stem cells after exposure to a high dosage of dexamethasone.
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高剂量地塞米松暴露后人骨髓间充质干细胞中长链非编码RNA和mRNA的差异表达谱
DOI：
10.1186/s13287-020-02040-8
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发表时间：
2021-01-06
影响因子：
7.5
通讯作者：
Jiang Y
中科院分区：
文献类型：
--
作者：
Li T;Xu Y;Wang Y;Jiang Y
背景人骨髓间充质干细胞（hBMSCs）的凋亡、细胞周期、增殖和分化异常，显著影响骨代谢和骨重建，导致各种骨骼疾病的发生。长期暴露于高剂量的地塞米松（Dex）可诱导间充质基质细胞（MSCs）凋亡并抑制其增殖，这可能是各种骨骼疾病的主要原因。方法采用Hoechst 33342/PI染色、流式细胞术、结晶紫法、β-半乳糖苷酶（β-GAL）活性测定、细胞凋亡率测定、细胞周期、增殖、衰老、成骨分化和成脂分化等方法，观察不同浓度Dex对hBMSCs的影响，并探讨其作用机制。进行茜素红S（ARS）染色测定和油红O（ORO）染色测定。使用微阵列分析来鉴定10− 6 mol/L Dex处理的hBMSCs中差异表达的lncRNA和mRNAs，并进行生物信息学分析以进一步探索这些差异表达的lncRNA和mRNAs在编码和非编码（CNC）网络中的作用。结果在10 - 8、10 - 7和10 - 6 mol/L浓度范围内，地塞米松可诱导hBMSCs凋亡，阻滞细胞周期，抑制成骨分化，促进成脂分化，且呈剂量依赖性。此外，10− 6 mol/L Dex明显诱导hBMSCs凋亡，抑制其增殖，并以时间依赖的方式增加其衰老。有趣的是，Dex对hBMSCs凋亡的这种时间依赖性作用在第7天达到平台，从第8天到第10天下降，而Dex处理的hBMSCs在第6天增加衰老。此外，微阵列分析鉴定了10− 6 mol/L Dex暴露后hBMSCs中总共137个差异表达的mRNA（90个上调，47个下调）和90个差异表达的lncRNA（61个上调，29个下调）。差异表达的mRNA和lncRNA与细胞凋亡、增殖和细胞周期的调节有关。同时，通过Path-Net分析中的相互作用网络，确定了mTOR信号通路、Ras信号通路、HIF-1信号通路、NF-κ B信号通路和TGF-β信号通路等参与这些过程的信号通路。此外，CNC网络进一步鉴定了78个参与细胞凋亡调控的核心调控基因。此外，qRT-PCR被用来确认的关键差异表达的mRNA和lncRNA的身份发现与细胞凋亡密切相关，以确认芯片dataset.ConclusionsIn总结，Dex对细胞凋亡，细胞周期，增殖，成骨分化和成脂分化的hBMSCs依赖于暴露时间和浓度的影响。10− 6 mol/L Dex持续作用7 d可能是诱导hBMSCs凋亡的适宜方案。在此方案下，差异表达的lncRNA和mRNA与细胞凋亡，细胞周期和增殖进行了鉴定，为进一步研究提供了一个新的研究方向。
BackgroundAbnormalities in apoptosis, cell cycle, proliferation, and differentiation of human bone marrow mesenchymal stem cells (hBMSCs) significantly impact bone metabolism and remodeling, resulting in various skeletal disorders. Long-term exposure to a high dosage of dexamethasone (Dex) induces apoptosis and inhibits the proliferation of mesenchymal stromal cells (MSCs), which are probable primary causes of various skeletal disorders. However, to date, the exact mechanisms of action of Dex on hBMSCs have not been fully elucidated.MethodsTo explore the effects of Dex on apoptosis, cell cycle, proliferation, senescence, osteogenic and adipogenic differentiation of hBMSCs at the various exposure times and concentrations, Hoechst 33342/PI staining, flow cytometry, crystal violet assay, β-galactosidase (β-GAL) activity assay, alizarin red S (ARS) staining assay, and Oil Red O (ORO) staining assay were performed. A microarray assay was used to identify differentially expressed lncRNAs and mRNAs in 10− 6mol/L Dex-treated hBMSCs, and a bioinformatics analysis was conducted to further explore the role of these differentially expressed lncRNAs and mRNAs in the coding and noncoding (CNC) network. Furthermore, the microarray results were validated using quantitative real-time PCR (qRT-PCR) analysis.ResultsOver the range of 10−8, 10−7, and 10−6mol/L, Dex induced apoptosis, arrest of the cell cycle, inhibition of osteogenic differentiation, and promotion adipogenic differentiation of the hBMSCs in a dose-dependent manner. In addition, 10−6mol/L Dex significantly induced apoptosis, suppressed proliferation, and increased the senescence of hBMSCs in a time-dependent manner. Interestingly, this time-dependent effect of Dex on the apoptosis of hBMSCs plateaued at the 7th day and decreased from the 8th day to the 10th day, while Dex treatment increased senescence of the hBMSCs on the 6th day. Furthermore, the microarray analysis identified a total of 137 differentially expressed mRNAs (90 upregulated and 47 downregulated) and 90 differentially expressed lncRNAs (61 upregulated and 29 downregulated) in hBMSCs after exposure to 10−6mol/L Dex. The differentially expressed mRNAs and lncRNAs were associated with the regulation of cell apoptosis, proliferation, and cell cycle. Meanwhile, several signaling pathways involved in these processes, including the mTOR signaling pathway, Ras signaling pathway, HIF-1 signaling pathway, NF-kappa B signaling pathway, and TGF-beta signaling pathway, also were identified through the interaction net in the significant pathways (Path-Net) analysis. Furthermore, the CNC network further identified 78 core regulatory genes involved in the regulation of apoptosis. Additionally, qRT-PCR was used to confirm the identity of the key differentially expressed mRNAs and lncRNAs found to be closely associated with cell apoptosis to confirm the reliability of the microarray dataset.ConclusionsIn summary, the effect of Dex on apoptosis, cell cycle, proliferation, and osteogenic differentiation and adipogenic differentiation of the hBMSCs depended on exposure time and concentration. Continuous exposure to 10−6mol/L of Dex for 7 days may be a suitable protocol for inducing the apoptosis of hBMSCs. Under this protocol, differentially expressed lncRNAs and mRNAs associated with apoptosis, cell cycle, and proliferation were identified, providing a new research direction for further studies.
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影响因子：
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作者：
Alessio N;Squillaro T;Özcan S;Di Bernardo G;Venditti M;Melone M;Peluso G;Galderisi U
通讯作者：
Galderisi U
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影响因子：
6.2
作者：
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影响因子：
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作者：
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DOI：
10.1006/bbrc.2001.6196
发表时间：
2002-01-11
影响因子：
3.1
作者：
Doi, M;Nagano, A;Nakamura, Y
通讯作者：
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影响因子：
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作者：
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通讯作者：
Wang, Tong