Zhang Shijie, Liu Yi, Zheng Zhong, Zeng Xuemin, Liu Dongxu, Wang Chunling, Ting Kang
Department of Orthodontics, Qilu Hospital of Shandong University, Jinan, China.
Shandong Provincial Key Laboratory of Oral Tissue Regeneration, School of Stomatology, Shandong University & Department of Orthodontics, School of Stomatology, Shandong University, Jinan, China.
Cell Physiol Biochem. 2018;47(2):667-679. doi: 10.1159/000490021. Epub 2018 May 22.
BACKGROUND/AIMS: In this study, we aimed to use bioinformatics tools to identify the specific miRNAs and mRNAs involved in osteogenic differentiation and to further explore the way in which miRNA regulates osteogenic differentiation.
The microarray GSE80614, which includes data from 3 human mesenchymal stromal cells (hMSCs) and 3 hMSCs after 72 hours (hr) of osteogenic differentiation, was used to screen for differentially expressed mRNAs. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of these mRNAs were conducted using Gene Set Enrichment Analysis (GSEA). Then, the miRanda website was employed to detect the binding sites of DHRS3. In vitro experiments, including RT-PCR and western blotting, were used to detect miR-233 and DHRS3 expression levels 7 and 14 days (d) after the induction of osteogenic differentiation using human bone marrow-derived mesenchymal stem cells (hBMSCs). The target relationship between miR-223 and DHRS3 was confirmed by a dual luciferase assay. ALP (alkaline phosphatase) staining, ARS (Alizarin Red S) staining and western blotting (Runx2, OPN, OCN) were used to detect the level of osteogenic differentiation after transfection with miR-223 mimics and DHRS3 cDNA.
In this study, 127 mRNAs differentially expressed during osteogenic differentiation were identified in GSE80614. GO term and KEGG pathway enrichment analyses found that the retinol metabolism pathway was activated during osteogenic differentiation and that DHRS3, which is involved in the pathway, was upregulated. During osteogenic differentiation in hBMSCs, miR-223 was gradually downregulated, while DHRS3 was upregulated. After 14 days of osteogenic differentiation, ALP and ARS staining assay results showed strong ALP activity and extracellular matrix calcification with the inhibition of miR-223 or the overexpression of DHRS3. Furthermore, the expression levels of Runx2, OPN, and OCN were upregulated with the knockdown of miR-223 or the overexpression of DHRS3, while the simultaneous transfection of a miR-223 agomir and DHRS3 cDNA resulted in no significant difference from the negative control (NC) group.
The inhibition of miR-223 promotes the osteogenic differentiation of hBMSCs via the upregulation of DHRS3.
背景/目的：在本研究中，我们旨在利用生物信息学工具鉴定参与成骨分化的特定微小RNA（miRNA）和信使核糖核酸（mRNA），并进一步探索miRNA调节成骨分化的方式。
使用包含3个人间充质基质细胞（hMSC）和成骨分化72小时后3个hMSC数据的基因芯片GSE80614筛选差异表达的mRNA。使用基因集富集分析（GSEA）对这些mRNA进行基因本体论（GO）和京都基因与基因组百科全书（KEGG）通路分析。然后，利用miRanda网站检测脱氢视黄醇还原酶3（DHRS3）的结合位点。在体外实验中，包括逆转录-聚合酶链反应（RT-PCR）和蛋白质免疫印迹法，用于检测用人骨髓间充质干细胞（hBMSC）诱导成骨分化7天和14天后miR-223和DHRS3的表达水平。通过双荧光素酶报告基因实验证实miR-223与DHRS3之间的靶向关系。使用碱性磷酸酶（ALP）染色、茜素红S（ARS）染色和蛋白质免疫印迹法（检测Runx2、骨桥蛋白（OPN）、骨钙素（OCN））检测转染miR-223模拟物和DHRS3 cDNA后的成骨分化水平。
在本研究中，在GSE80614中鉴定出127个在成骨分化过程中差异表达的mRNA。GO术语和KEGG通路富集分析发现，视黄醇代谢途径在成骨分化过程中被激活，参与该途径的DHRS3上调。在hBMSC成骨分化过程中，miR-223逐渐下调，而DHRS3上调。成骨分化14天后，ALP和ARS染色分析结果显示，抑制miR-223或过表达DHRS3时，ALP活性增强且细胞外基质钙化。此外，Runx2、OPN和OCN的表达水平在miR-223敲低或DHRS3过表达时上调，而同时转染miR-223激动剂和DHRS3 cDNA与阴性对照（NC）组相比无显著差异。
抑制miR-223通过上调DHRS3促进hBMSC的成骨分化。
