Puerarin Suppresses the Self-Renewal of Murine Embryonic Stem Cells by Inhibition of REST-MiR-21 Regulatory Pathway.
OBJECTIVE
Puerarin shows a wide range of biological activities, including affecting the cardiac differentiation from murine embryonic stem (mES) cells. However, little is known about its effect and mechanism of action on the self-renewal of mES cells. This study aimed to determine the effect of puerarin on the self-renewal and pluripotency of mES cells and its underlying mechanisms.


METHODS
RT-PCR and real-time PCR were used to detect the transcripts of core transcription factors, specific markers for multiple lineages, REST and microRNA-21 (miR-21). Colony-forming assay was performed to estimate the self-renewal capacity of mES cells. Western blotting and wortmannin were employed to explore the role of PI3K/Akt signaling pathway in the inhibitory action of puerarin on REST transcript. Transfected mES cells with antagomir21 were used to confirm the role of miR-21 in the action of puerarin on cell self-renewal.


RESULTS
Puerarin significantly decreased the percentage of the self-renewal colonies, and suppressed the transcripts of Oct4, Nanog, Sox2, c-Myc and REST. Besides, PECAM, NCAM and miR-21 were up-regulated both under the self-renewal conditions and at day 4 of differentiation. The PI3K inhibitor wortmannin successfully reversed the mRNA expression changes of REST, Nanog and Sox2. Transfection of antagomir21 efficiently reversed the effects of puerarin on mES cells self-renewal.


CONCLUSIONS
Inhibition of REST-miR-21 regulatory pathway may be the key mechanism of puerarin-induced suppression of mES cells self-renewal.
Mengmeng
M
Yin
Yin
Y
Yuan
Yurong
Y
Cui
Xian
X
Hong
Hongyan
H
Luo
Xinwu
X
Hu
Ming
M
Tang
Jurgen
J
Hescheler
Jiaoya
J
Xi
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D000066673:Cell Self Renewal / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D007529:Isoflavones / Q000494:pharmacology*; D051379:Mice; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014665:Vasodilator Agents / Q000494:pharmacology*; D000077191:Wortmannin; D000099229:RE1-Silencing Transcription Factor
D000730:Androstadienes; D007529:Isoflavones; D035683:MicroRNAs; D012097:Repressor Proteins; D014665:Vasodilator Agents; D000077191:Wortmannin; D000099229:RE1-Silencing Transcription Factor; C531950:MIRN21 microRNA, mouse; C033607:puerarin