Background
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
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.
Methods
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.
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.