Department of Pharmaceutical Sciences, College of Pharmacy and Pharmaceutical Sciences, Washington State University, Spokane, WA, United States of America.
Department of Medicine, Rheumatology, University of Washington, Seattle, WA, United States of America.
PLoS One. 2020 Jun 30;15(6):e0225044. doi: 10.1371/journal.pone.0225044. eCollection 2020.
Cellular stress response mechanisms normally function to enhance survival and allow for cells to return to homeostasis following an adverse event. Cancer cells often co-opt these same mechanisms as a means to evade apoptosis and mitigate a state of constant cellular stress. Activating transcription factor 5 (ATF5) is upregulated under diverse stress conditions and is overexpressed in a variety of cancers. It was demonstrated ATF5 is a survival factor in transformed, but not normal cells. However, the regulation of ATF5 is not fully understood. The purpose of the present study was to investigate miRNA regulation at the 3' untranslated region (UTR) of ATF5, with the goal of demonstrating a reversal of the upregulation of ATF5 induced under diverse cellular stress in cancer cells. A multifactorial approach using in silico analysis was employed to identify miRNAs 433-3p, 520b-3p, and 129-5p as potential regulators of ATF5, based on their predicted binding sites over the span of the ATF5 3' UTR. Luciferase reporter assay data validated all three miRNA candidates by demonstrating direct binding to the target ATF5 3'. However, functional studies revealed miR-520b-3p as the sole candidate able to reverse the upregulation of ATF5 protein under diverse cellular stress. Additionally, miR-520b-3p levels were inversely related to ATF5 mRNA under endoplasmic reticulum stress and amino acid deprivation. This is the first evidence that regulation at the 3' UTR is involved in modulating ATF5 levels under cellular stress and suggests an important role for miRNA-520b-3p in the regulation of ATF5.
细胞应激反应机制通常可增强生存能力,并使细胞在不利事件后恢复到体内平衡。癌细胞经常采用相同的机制来逃避细胞凋亡并减轻持续的细胞应激状态。激活转录因子 5(ATF5)在各种应激条件下上调,并在多种癌症中过表达。已经证明,ATF5 是转化细胞而不是正常细胞的存活因子。然而,ATF5 的调节尚未完全阐明。本研究的目的是研究 ATF5 的 3'非翻译区(UTR)的 miRNA 调节,目标是证明在癌细胞中各种细胞应激下诱导的 ATF5 上调得到逆转。采用多因素方法进行计算机分析,根据 ATF5 3'UTR 上的预测结合位点,鉴定出 miRNA 433-3p、520b-3p 和 129-5p 作为 ATF5 的潜在调节剂。荧光素酶报告基因测定数据通过证明这三种 miRNA 候选物与靶 ATF5 3'直接结合,验证了所有三种 miRNA 候选物。然而,功能研究表明,miR-520b-3p 是唯一能够在各种细胞应激下逆转 ATF5 蛋白上调的候选物。此外,在内质网应激和氨基酸剥夺下,miR-520b-3p 的水平与 ATF5 mRNA 呈负相关。这是第一个表明 3'UTR 调节参与调节细胞应激下 ATF5 水平的证据,并表明 miRNA-520b-3p 在 ATF5 调节中起重要作用。