Bioengineering Laboratory, Department of Mechanical Engineering and Louisiana State University, Baton Rouge, Louisiana, USA.
Department of Biological and Agricultural Engineering, Louisiana State University, Baton Rouge, Louisiana, USA.
Stem Cells Dev. 2021 Mar;30(5):265-288. doi: 10.1089/scd.2020.0173. Epub 2021 Feb 8.
While several microRNAs (miRNAs) that regulate the endotheliogenesis and further promote angiogenesis have been identified in various cancers, the identification of miRNAs that can drive the differentiation of adipose derived stromal/stem cells (ASCs) into the endothelial lineage has been largely unexplored. In this study, CD34+ ASCs sorted using magnetic bead separation were induced to differentiate along the endothelial pathway. miRNA sequencing of ASCs at day 3, 9, and 14 of endothelial differentiation was performed on Ion Proton sequencing system. The data obtained by this high-throughput method were aligned to the human genome HG38, and the differentially expressed miRNAs during endothelial differentiation at various time points (day 3, 9, and 14) were identified. The gene targets of the identified miRNAs were obtained through miRWalk database. The network-pathway analysis of miRNAs and their targets was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatic tools to determine the potential candidate miRNAs that promote endothelial differentiation. Based on these analyses, six upregulated miRNAs (miR-181a-5p, miR-330-5p, miR-335-3p, miR-15b-5p, miR-99a-5p, and miR-199a-5p) and six downregulated miRNAs (miR-145-5p, miR-155-5p, miR-193a-3p, miR-125a-5p, miR-221-5p, and miR-222-3p) were chosen for further studies. In vitro evaluation of these miRNAs to induce endothelial differentiation when transfected into CD34+ sorted ASCs was studied using Von Willebrand Factor (VWF) staining and quantitative real time-polymerase chain reaction (qRT-PCR). Our results suggest that miRNAs: 335-5p, 330-5p, 181a-5p and anti-miRNAs: 125a-5p, 145-5p can likely induce endothelial differentiation in CD34+ sorted ASCs. Further studies are clearly required to elucidate the specific mechanisms on how miRNAs or anti-miRNAs identified through bioinformatics approach can induce the endotheliogenesis in ASCs.
虽然已经在各种癌症中鉴定出了几种调节血管生成的内皮发生并进一步促进血管生成的 microRNA（miRNA），但鉴定可以驱动脂肪来源的基质/干细胞（ASC）分化为内皮谱系的 miRNA 在很大程度上仍未得到探索。在这项研究中，使用磁珠分离分选 CD34+ ASCs 以沿内皮途径诱导分化。在 Ion Proton 测序系统上对第 3、9 和 14 天内皮分化的 ASCs 进行 miRNA 测序。通过这种高通量方法获得的数据与人类基因组 HG38 对齐，并鉴定了在不同时间点（第 3、9 和 14 天）内皮分化过程中差异表达的 miRNA。通过 miRWalk 数据库获得鉴定 miRNA 的靶基因。使用数据库注释、可视化和综合发现（DAVID）生物信息学工具对 miRNA 及其靶标的网络途径分析，以确定促进内皮分化的潜在候选 miRNA。基于这些分析，选择了六个上调的 miRNA（miR-181a-5p、miR-330-5p、miR-335-3p、miR-15b-5p、miR-99a-5p 和 miR-199a-5p）和六个下调的 miRNA（miR-145-5p、miR-155-5p、miR-193a-3p、miR-125a-5p、miR-221-5p 和 miR-222-3p）用于进一步研究。通过 Von Willebrand 因子（VWF）染色和定量实时聚合酶链反应（qRT-PCR）研究转染到 CD34+分选的 ASC 中这些 miRNA 诱导内皮分化的体外评估。我们的结果表明，miRNA：335-5p、330-5p、181a-5p 和 anti-miRNA：125a-5p、145-5p 可能诱导 CD34+分选的 ASC 内皮分化。显然需要进一步研究来阐明通过生物信息学方法鉴定的 miRNA 或 anti-miRNA 如何诱导 ASC 中的内皮发生的具体机制。
