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微小 RNA-32 沉默 WWP2 表达以维持人羊膜上皮干细胞的多能性和β胰岛样细胞分化。

MicroRNA‑32 silences WWP2 expression to maintain the pluripotency of human amniotic epithelial stem cells and β islet‑like cell differentiation.

机构信息

Department of Obstetrics, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai 200040, P.R. China.

Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, P.R. China.

出版信息

Int J Mol Med. 2018 Apr;41(4):1983-1991. doi: 10.3892/ijmm.2018.3436. Epub 2018 Jan 29.

Abstract

Human amniotic epithelial stem cells (HuAECs) exhibit pluripotent characteristics, which are similar to those of embryonic stem cells, and can differentiate into various adult tissues and cells through directed induction. However, in culture, HuAECs tend to lose their pluripotency, and their directed differentiation capability declines with increasing passage number. The stem cell pluripotency factor octamer‑binding protein 4 (Oct4) is an important transcription factor that promotes stem cell self‑proliferation and maintains their pluripotency. Previous studies have demonstrated that WW domain containing E3 ubiquitin protein ligase 2 (WWP2) negatively regulates Oct4 expression and stem cell pluripotency. Therefore, the present study aimed to investigate the regulation of WWP2 by microRNAs (miRs), and to evaluate the expression of the downstream factor Oct4 and the maintenance of HuAEC pluripotency. Bioinformatics analysis identified a complementary binding site for miR‑32 in the 3'untranslated region of the WWP2 gene, thus suggesting that it may be a target gene of miR‑32. Post‑infection of HuAECs with a vector overexpressing miR‑32, the endogenous expression of WWP2 was significantly decreased, whereas Oct4 expression was significantly increased. Furthermore, miR‑32‑infected cells differentiated into β islet‑like cells by directed induction. The results indicated that after induction, HuAECs overexpressing miR‑32 also overexpressed the biomarkers of β islet‑like cells. In addition, the ability to secrete insulin was markedly enhanced in response to glucose stimulation, in cells overexpressing miR‑32. In conclusion, the present study suggested that miR‑32 may effectively inhibit WWP2 expression in HuAECs and promote Oct4 overexpression to maintain their pluripotency.

摘要

人羊膜上皮干细胞(HuAECs)表现出多能性特征,类似于胚胎干细胞,并且可以通过定向诱导分化为各种成体组织和细胞。然而,在培养过程中,HuAECs 往往会失去多能性,并且其定向分化能力随着传代次数的增加而下降。多能性转录因子八聚体结合蛋白 4(Oct4)是一种重要的转录因子,可促进干细胞自我增殖并维持其多能性。先前的研究表明,WW 结构域包含 E3 泛素蛋白连接酶 2(WWP2)负调控 Oct4 表达和干细胞多能性。因此,本研究旨在探讨 microRNAs(miRs)对 WWP2 的调控作用,并评估下游因子 Oct4 的表达和 HuAEC 多能性的维持。生物信息学分析表明,miR-32 在 WWP2 基因的 3'非翻译区存在互补结合位点,提示其可能是 miR-32 的靶基因。HuAECs 感染过表达 miR-32 的载体后,WWP2 的内源性表达明显降低,而 Oct4 的表达明显增加。此外,经定向诱导,miR-32 感染的细胞分化为胰岛样细胞。结果表明,诱导后过表达 miR-32 的 HuAECs 也过表达了胰岛样细胞的标志物。此外,过表达 miR-32 的细胞在葡萄糖刺激下胰岛素分泌能力显著增强。综上所述,本研究表明 miR-32 可能通过有效抑制 HuAECs 中 WWP2 的表达并促进 Oct4 的过表达来维持其多能性。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4939/5810217/fbd72ed0bfd7/IJMM-41-04-1983-g00.jpg

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