Fráguas Mariane Serra, Eggenschwiler Reto, Hoepfner Jeannine, Schiavinato Josiane Lilian Dos Santos, Haddad Rodrigo, Oliveira Lucila Habib Bourguignon, Araújo Amélia Góes, Zago Marco Antônio, Panepucci Rodrigo Alexandre, Cantz Tobias
Department of Clinical Medicine, Faculty of Medicine, University of São Paulo (FMRP-USP), Brazil; National Institute of Science and Technology in Stem Cell and Cell Therapy (INCTC), Center for Cell Therapy (CTC), Regional Blood Center, Ribeirão Preto, Brazil; Translational Hepatology and Stem Cell Biology, REBIRTH Cluster of Excellence and Dept. of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Hannover, Germany.
Translational Hepatology and Stem Cell Biology, REBIRTH Cluster of Excellence and Dept. of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Hannover, Germany.
Stem Cell Res. 2017 Mar;19:21-30. doi: 10.1016/j.scr.2016.12.020. Epub 2016 Dec 19.
Somatic cell reprogramming by transcription factors and other modifiers such as microRNAs has opened broad avenues for the study of developmental processes, cell fate determination, and interplay of molecular mechanisms in signaling pathways. However, many of the mechanisms that drive nuclear reprogramming itself remain yet to be elucidated. Here, we analyzed the role of miR-29 during reprogramming in more detail. Therefore, we evaluated miR-29 expression during reprogramming of fibroblasts transduced with lentiviral OKS and OKSM vectors and we show that addition of c-MYC to the reprogramming factor cocktail decreases miR-29 expression levels. Moreover, we found that transfection of pre-miR-29a strongly decreased OKS-induced formation of GFP-colonies in MEF-cells from Oct4-eGFP reporter mouse, whereas anti-miR-29a showed the opposite effect. Furthermore, we studied components of two pathways which are important for reprogramming and which involve miR-29 targets: active DNA-demethylation and Wnt-signaling. We show that inhibition of Tet1, Tet2 and Tet3 as well as activation of Wnt-signaling leads to decreased reprogramming efficiency. Moreover, transfection of pre-miR-29 resulted in elevated expression of β-Catenin transcriptional target sFRP2 and increased TCF/LEF-promoter activity. Finally, we report that Gsk3-β is a direct target of miR-29 in MEF-cells. Together, our findings contribute to the understanding of the molecular mechanisms by which miR-29 influences reprogramming.
通过转录因子和其他修饰因子（如微小RNA）进行的体细胞重编程为发育过程、细胞命运决定以及信号通路中分子机制的相互作用研究开辟了广阔的道路。然而，驱动核重编程本身的许多机制仍有待阐明。在此，我们更详细地分析了miR - 29在重编程过程中的作用。因此，我们评估了用慢病毒OKS和OKSM载体转导的成纤维细胞重编程过程中miR - 29的表达，结果表明在重编程因子混合物中添加c - MYC会降低miR - 29的表达水平。此外，我们发现转染pre - miR - 29a会强烈降低来自Oct4 - eGFP报告基因小鼠的MEF细胞中OKS诱导的GFP集落形成，而抗miR - 29a则显示出相反的效果。此外，我们研究了对重编程很重要且涉及miR - 29靶标的两条信号通路的组成部分：活性DNA去甲基化和Wnt信号通路。我们发现抑制Tet1、Tet2和Tet3以及激活Wnt信号通路会导致重编程效率降低。此外，转染pre - miR - 29会导致β - 连环蛋白转录靶标sFRP2的表达升高以及TCF/LEF启动子活性增加。最后，我们报告Gsk3 - β是MEF细胞中miR - 29的直接靶标。总之，我们的研究结果有助于理解miR - 29影响重编程的分子机制。
