Dai Yujian, Li Jinshan, Li Mingyang, Liu Zhihui, Liu Jiao, An Liyou, Du Fuliang
Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University Nanjing 210046, PR China.
Renova Life, Inc. College Park, Maryland 20742, USA.
Am J Transl Res. 2020 Dec 15;12(12):8147-8161. eCollection 2020.
Methyl-CpG-binding domain 3 (Mbd3) is a core repressor complex component. Although Mbd3 is required for the pluripotency of embryonic stem cells (ES), the role of Mbd3 in mouse ES (mES) cell apoptosis remains undefined. In this study naïve-state mES were derived and maintained in the presence of a selective protein kinase C pathway inhibitor (PKCi; Gӧ6983) to study the function of Mbd3 during mES apoptosis. Mbd3 overexpression in mES decreased the total cell number and viability, and it also dramatically increased the rate of apoptosis. Further investigation of Mbd3 overexpression revealed a 3-fold increase in the proapoptotic/prosurvival protein ratio (Bax/Bcl-2) and elevated RNA expression levels of apoptosis-related genes, including , and , with reduced RNA expression levels. Removal of PKCi from the mES cell culture resulted in upregulated Mbd3 expression and apoptosis, similar to the effects of Mbd3 overexpression. Furthermore, specific knockdown of endogenous Mbd3 partially rescued the mES apoptosis induced by the removal of PKCi, thus increasing the total cell number and viability while decreasing the rate of apoptosis. Additionally, , and RNA expression levels were partially reduced, and that of was partially increased. Our findings support Mbd3 as a pivotal regulator of apoptosis in mES.
甲基化CpG结合结构域3（Mbd3）是一种核心阻遏复合物成分。尽管Mbd3是胚胎干细胞（ES）多能性所必需的，但Mbd3在小鼠胚胎干细胞（mES）凋亡中的作用仍不明确。在本研究中，在选择性蛋白激酶C途径抑制剂（PKCi；Gӧ6983）存在的情况下获得并维持了原始状态的mES，以研究Mbd3在mES凋亡过程中的功能。mES中Mbd3的过表达降低了总细胞数和活力，并且还显著增加了凋亡率。对Mbd3过表达的进一步研究显示，促凋亡/抗生存蛋白比率（Bax/Bcl-2）增加了3倍，凋亡相关基因的RNA表达水平升高，包括 、 和 ，而 RNA表达水平降低。从mES细胞培养物中去除PKCi导致Mbd3表达上调和凋亡，类似于Mbd3过表达的效果。此外，内源性Mbd3的特异性敲低部分挽救了因去除PKCi诱导的mES凋亡，从而增加了总细胞数和活力，同时降低了凋亡率。此外， 、 和 RNA表达水平部分降低，而 的表达水平部分升高。我们的研究结果支持Mbd3作为mES凋亡的关键调节因子。
