摘要
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. Methods: 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. Results: 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. Conclusion: The inhibition of miR-223 promotes the osteogenic differentiation of hBMSCs via the upregulation of DHRS3.
摘要译文
背景/目的：在这项研究中，我们的目标是使用生物信息学工具来识别与成骨分化有关的特定miRNA和mRNA，并进一步探索miRNA调节成骨分化的方式。方法：微阵列GSE80614，其包括来自3个人间充质基质细胞（hMSC）和3个hMSC的成骨分化72小时（hr）后的数据，用于筛选差异表达的mRNA。使用基因集富集分析（GSEA）进行这些mRNA的基因本体论（GO）和京都百科全书基因和基因组（KEGG）途径分析。然后，miRanda网站用于检测DHRS3的结合位点。体外实验，包括RT-PCR和蛋白质印迹，用人骨髓来源的间充质干细胞（hBMSCs）诱导成骨分化后7天和14天（d）用于检测miR-233和DHRS3表达水平。通过双荧光素酶测定证实miR-223和DHRS3之间的靶关系。 ALP（碱性磷酸酶）染色，ARS（茜素红S）染色和蛋白质印迹（Runx2，OPN，OCN）用于检测用miR-223模拟物和DHRS3 cDNA转染后的成骨分化水平。结果：在这项研究中，在GSE80614中鉴定了在成骨分化期间差异表达的127种mRNA。GO术语和KEGG途径富集分析发现视黄醇代谢途径在成骨分化期间被激活，并且DHRS3参与该途径，被上调了。在hBMSCs的成骨分化期间，miR-223逐渐下调，而DHRS3上调。成骨分化14天后，ALP和ARS染色测定结果显示强ALP活性和细胞外基质钙化，抑制miR-223或DHRS3的过表达。此外，随着miR-223的敲低或DHRS3的过表达，Runx2，OPN和OCN的表达水平上调，同时转染miR-223 agomir和DHRS3 cDNA导致与阴性对照（NC）组无显着差异。结论：miR-223的抑制通过上调DHRS3促进hBMSCs的成骨分化。
Zhang S. ; Liu Y. ; Zheng Z. ; Zeng X. ; Liu D. ; Wang C. ; Ting K.. MicroRNA-223 Suppresses Osteoblast Differentiation by Inhibiting DHRS3[J]. Cellular Physiology and Biochemistry, 2018,47(2)