Yin Mengmeng, Yuan Yin, Cui Yurong, Hong Xian, Luo Hongyan, Hu Xinwu, Tang Ming, Hescheler Jurgen, Xi Jiaoya
Cell Physiol Biochem. 2015;37(2):527-36. doi: 10.1159/000430374.
BACKGROUND/AIMS: 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.
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.
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.
Inhibition of REST-miR-21 regulatory pathway may be the key mechanism of puerarin-induced suppression of mES cells self-renewal.
背景/目的：葛根素具有广泛的生物学活性，包括影响小鼠胚胎干细胞（mES细胞）的心脏分化。然而，关于其对mES细胞自我更新的作用及其作用机制知之甚少。本研究旨在确定葛根素对mES细胞自我更新和多能性的影响及其潜在机制。
采用RT-PCR和实时PCR检测核心转录因子、多谱系特异性标志物、REST和微小RNA-21（miR-21）的转录本。进行集落形成试验以评估mES细胞的自我更新能力。采用蛋白质免疫印迹法和渥曼青霉素探讨PI3K/Akt信号通路在葛根素对REST转录本抑制作用中的作用。用抗miR-21转染mES细胞以证实miR-21在葛根素对细胞自我更新作用中的作用。
葛根素显著降低自我更新集落的百分比，并抑制Oct4、Nanog、Sox2、c-Myc和REST的转录本。此外，在自我更新条件下和分化第4天时，PECAM、NCAM和miR-21均上调。PI3K抑制剂渥曼青霉素成功逆转了REST、Nanog和Sox2的mRNA表达变化。抗miR-21转染有效逆转了葛根素对mES细胞自我更新的影响。
抑制REST-miR-21调控通路可能是葛根素诱导mES细胞自我更新受抑制的关键机制。
