Department of Cardiology, Southwest Hospital, Third Military Medical University (Army Medical University), China.
Neuroscience Division, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia.
Biosci Rep. 2019 Feb 26;39(2). doi: 10.1042/BSR20181341. Print 2019 Feb 28.
Geminin is an inhibitor of DNA replication licensing and cell cycle. Our previous study demonstrates that Geminin plays an important role in regulating phenotypic diversity and growth of vascular smooth cells (VSMCs). Ubiquitin-like with PHD and RING Finger domains 1 (UHRF1) is an epigenetic coordinator, whose RING domain confers intrinsic E3 ligase activity, mediating the ubiquitination of several proteins and the protein-protein interaction. Aberrant expression of UHRF1 was related to aggressiveness of multiple human malignancies, where knockdown of UHRF1 led to decreased proliferation of cancer cells. However, it is unclear whether proper UHRF1 function is involved in aberrant proliferation and phenotypic switching of VSMCs via altering Geminin protein levels. In present study, in UHRF1-overexpressing A10 cells, 3H-thymidine and 5-ethynyl-20-deoxyuridine (EdU) and CCK8 were used to examine the proliferation of VSMCs. RT-PCR and Western blot analyses were performed to investigate whether UHRF1-mediated effects were achieved by altering Geminin expression in VSMCs. RNA-seq analysis was performed to dissect related mechanisms or signaling pathways of these effects. The results of experiments suggested that UHRF1 prompted proliferation and cell cycle of VSMCs via the down-regulation of Geminin protein levels with no change in Geminin mRNA expression. Besides, PI3K-Akt signaling pathway was increased upon UHRF1 up-regulation. Our study demonstrated that overexpressing UHRF1 was involved in VSMCs proliferation through reducing inhibitory Geminin protein levels to promote cell cycle as well as activating PI3K-Akt signaling. This may provide key knowledge for the development of better strategies to prevent diseases related to VSMCs abnormal proliferation.
增殖细胞核抗原是 DNA 复制起始点识别复合物的抑制因子，也是细胞周期的调控因子。我们之前的研究表明增殖细胞核抗原在调节血管平滑肌细胞（VSMCs）的表型多样性和生长中发挥着重要作用。泛素样含 PHD 和 RING 指结构域蛋白 1（UHRF1）是一种表观遗传协调因子，其 RING 结构域赋予内在的 E3 连接酶活性，介导几种蛋白质的泛素化和蛋白质-蛋白质相互作用。UHRF1 的异常表达与多种人类恶性肿瘤的侵袭性有关，其中 UHRF1 的敲低导致癌细胞增殖减少。然而，UHRF1 的适当功能是否通过改变 Geminin 蛋白水平参与 VSMCs 的异常增殖和表型转换尚不清楚。在本研究中，在 UHRF1 过表达的 A10 细胞中，使用 3H-胸腺嘧啶和 5-乙炔基-20-脱氧尿苷（EdU）和 CCK8 检测 VSMCs 的增殖。进行 RT-PCR 和 Western blot 分析以研究 UHRF1 介导的效应是否通过改变 VSMCs 中的 Geminin 表达来实现。进行 RNA-seq 分析以剖析这些效应的相关机制或信号通路。实验结果表明，UHRF1 通过下调 Geminin 蛋白水平而不改变 Geminin mRNA 表达来促进 VSMCs 的增殖和细胞周期。此外，PI3K-Akt 信号通路在 UHRF1 上调时增加。我们的研究表明，过表达 UHRF1 通过降低抑制性 Geminin 蛋白水平来促进细胞周期，从而参与 VSMCs 的增殖，以及激活 PI3K-Akt 信号。这可能为预防与 VSMCs 异常增殖相关的疾病提供关键知识。
