MicroRNA-302a is involved in folate deficiency-induced apoptosis through the AKT-FOXO1-BIM pathway in mouse embryonic st
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RESEARCH
MicroRNA‑302a is involved in folate deficiency‑induced apoptosis through the AKT‑FOXO1‑BIM pathway in mouse embryonic stem cells Yan Liang1, Dingding Cao2, Yuanyuan Li2, Zhuo Liu2 and Jianxin Wu2*
Abstract Background: Our previous study had shown that microRNA (miR)-302a played a key role in folate deficiencyinduced 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. Methods: The TF-miRNA gene regulation network was constructed via bioinformatics methods. Chromatin immunocoprecipitation 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. Results: 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. Conclusion: 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. Keywords: miR-302a, Akt/FOXO1/Bim pathway, Folate deficiency, Cell apoptosis Introduction Folate is a water-soluble vitamin and a cofactor for the transportation of one-carbon units in DNA biosynthesis, as well as in hundreds of methylation reactions [1, 2]. Few studies investigated whether and how folate deficiency *Correspondence: [email protected] 2 Department of Biochemistry and Immunology, Capital Institute of Pediatrics, Beijing 100020, China Full list of author information is available at the end of the article
affects the normal development of a growing embryo [3]. The expression of micro RNA (miRNA) can be regulated by epigenetic mechanisms, such as DNA methylation or histone modifications [4]. Regulatory transcription factors (TFs) control miRNA expression, working at the level of transcription initiation by directly binding to genomic DNA targets, resulting in miRNA activation or repression. TFs and miRNAs are two key elements in gene regulation. The relationships between TFs, miRNAs, and target genes are extremely complex and play
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