Our previous study had shown that microRNA (miR)-302a played a key role in folate deficiency-induced apoptosis in mouse embryonic stem cells. However, details regarding the mechanism remain unclear. Transcription factors (TFs) and miRNAs are two key elements in gene regulation. The aim of this study is to construct the TF-miRNA gene regulation network and demonstrate its possible mechanism. The TF-miRNA gene regulation network was constructed via bioinformatics methods. Chromatin immuno-coprecipitation PCR was selected to confirm the binding between miR-302a and TF. mRNA and protein levels were detected by Real-time quantitative PCR and western blotting. TargetScan prediction and Dual-Luciferase Reporter Assay system were used to confirm whether the miRNA binded directly to the predicted target gene. FOXO1 and miR-302a were selected as the key TF and miRNA, respectively. FOXO1 was confirmed to bind directly to the upstream promoter region of miR-302a. Real-time quantitative PCR and immunoblotting showed that in folate-free conditions, miR-302a and AKT were down regulated, while FOXO1 and Bim were up-regulated significantly. Additionally, treatment with LY294002 inhibitor revealed the involvement of the Akt/FOXO1/Bim signaling pathway in folate deficiency-induced apoptosis, rather than the ERK pathway. Finally, TargetScan prediction and double luciferase reporting experiments illustrated the ability of miR-302a to target the Bim 3′UTR region. The involvement of miR-302a in folate deficiency-induced apoptosis through the AKT-FOXO1-BIM pathway in mESCs is a unique demonstration of the regulation mechanism of nutrient expression in embryonic development.

中文翻译：

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MicroRNA-302a通过小鼠胚胎干细胞中的AKT-FOXO1-BIM途径参与叶酸缺乏诱导的细胞凋亡

我们以前的研究表明，microRNA（miR）-302a在叶酸缺乏诱导的小鼠胚胎干细胞凋亡中起着关键作用。但是，有关该机制的细节仍不清楚。转录因子（TFs）和miRNA是基因调控中的两个关键要素。这项研究的目的是建立TF-miRNA基因调控网络，并证明其可能的机制。TF-miRNA基因调控网络是通过生物信息学方法构建的。选择染色质免疫共沉淀PCR以确认miR-302a和TF之间的结合。通过实时定量PCR和蛋白质印迹检测mRNA和蛋白质水平。使用TargetScan预测和Dual-Luciferase Reporter Assay系统确认miRNA是否直接与预测的靶基因结合。FOXO1和miR-302a分别被选为关键TF和miRNA。证实FOXO1直接结合到miR-302a的上游启动子区域。实时定量PCR和免疫印迹显示，在无叶酸条件下，miR-302a和AKT被下调，而FOXO1和Bim被显着上调。此外，用LY294002抑制剂治疗显示Akt / FOXO1 / Bim信号通路参与叶酸缺乏诱导的细胞凋亡，而不是ERK通路。最后，TargetScan预测和双重荧光素酶报告实验说明了miR-302a靶向Bim 3'UTR区域的能力。