Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia; Genetics and Regenerative Medicine Research Centre, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
Gene. 2019 May 20;697:201-212. doi: 10.1016/j.gene.2019.02.014. Epub 2019 Feb 12.
MicroRNA-3099 is highly expressed during neuronal differentiation and development of the central nervous system. Here we characterised the role of miR-3099 during neural differentiation and embryonic brain development using a stable and regulatable mouse embryonic stem cell culture system for miR-3099 expression and in utero electroporation of miR-3099 expression construct into E15.5 embryonic mouse brains. In the in vitro system, miR-3099 overexpression upregulated gene related to neuronal markers such as Tuj1, NeuN, Gat1, vGluT1 and vGluT2. In contrast, gene related to astrocyte markers (Gfap, S100β and Slc1a3) were suppressed upon overexpression of miR-3099. Furthermore, miR-3099 overexpression between E15.5 and E18.5 mouse embryonic brains led to disorganised neuronal migration potentially due to significantly decreased Gfap+ cells. Collectively, our results indicated that miR-3099 plays a role in modulating and regulating expression of key markers involved in neuronal differentiation. In silico analysis was also performed to identify miR-3099 homologues in the human genome, and candidates were validated by stem-loop RT-qPCR. Analysis of the miR-3099 seed sequence AGGCUA against human transcriptomes revealed that a potential miRNA, mds21 (Chr21:39186698-39186677) (GenBank accession ID: MK521584), was 100% identical to the miR-3099 seed sequence. Mds21 expression was observed and validated in various human cell lines (293FT, human Wharton's jelly and dental pulp mesenchymal stem cells, and MCF-7, MDA-MB-231, C-Sert, SW780, RT112, 5637, EJ28 and SH-SY5Y cells), with the highest levels detected in human mesenchymal stem cell lines. The analysis validated mds21 as a novel miRNA and a novel homologue of miR-3099 in the human genome.
miR-3099 在神经元分化和中枢神经系统发育过程中高度表达。在这里，我们使用稳定和可调节的小鼠胚胎干细胞培养系统来表达 miR-3099，以及在 E15.5 胚胎小鼠脑中进行 miR-3099 表达构建体的体内电穿孔，来研究 miR-3099 在神经分化和胚胎大脑发育中的作用。在体外系统中，miR-3099 的过表达上调了与神经元标记物相关的基因，如 Tuj1、NeuN、Gat1、vGluT1 和 vGluT2。相比之下，miR-3099 的过表达抑制了与星形胶质细胞标记物（Gfap、S100β 和 Slc1a3）相关的基因。此外，在 E15.5 和 E18.5 小鼠胚胎脑中过表达 miR-3099 导致神经元迁移紊乱，可能是由于 Gfap+细胞显著减少所致。总之，我们的结果表明，miR-3099 在调节神经元分化过程中关键标记物的表达中发挥作用。还进行了计算机分析以鉴定人类基因组中的 miR-3099 同源物，并通过茎环 RT-qPCR 对候选物进行了验证。分析 miR-3099 种子序列 AGGCUA 对人类转录组的影响表明，一种潜在的 miRNA，mds21（Chr21:39186698-39186677）（GenBank 登录号：MK521584），与 miR-3099 种子序列完全相同。在各种人类细胞系（293FT、人 Wharton's 果冻和牙髓间充质干细胞以及 MCF-7、MDA-MB-231、C-Sert、SW780、RT112、5637、EJ28 和 SH-SY5Y 细胞）中观察到并验证了 mds21 的表达，其中在人间充质干细胞系中检测到最高水平。该分析验证了 mds21 是人类基因组中一种新的 miRNA 和 miR-3099 的新同源物。
