Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii, USA; Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, Hawaii, USA.
Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii, USA.
J Biol Chem. 2022 Mar;298(3):101634. doi: 10.1016/j.jbc.2022.101634. Epub 2022 Jan 25.
While miRs have been extensively studied in the context of malignancy and tumor progression, their functions in regulating T-cell activation are less clear. In initial studies, we found reduced levels of miR-15a/16 at 3 to 18 h post-T-cell receptor (TCR) stimulation, suggesting a role for decreased levels of this miR pair in shaping T-cell activation. To further explore this, we developed an inducible miR15a/16 transgenic mouse model to determine how elevating miR-15a/16 levels during early stages of activation would affect T-cell proliferation and to identify TCR signaling pathways regulated by this miR pair. Doxycycline (DOX)-induced expression of miR-15a/16 from 0 to 18 h post-TCR stimulation decreased ex vivo T-cell proliferation as well as in vivo antigen-specific T-cell proliferation. We also combined bioinformatics and proteomics approaches to identify the mitogen-activated protein kinase kinase 1 (MEK1) (Map2k1) as a target of miR-15a/16. MEK1 targeting by miR-15a/16 was confirmed using miR mimics that decreased Map2k1 mRNA containing the 3'-UTR target nucleotide sequence (UGCUGCUA) but did not decrease Map2k1 containing a mutated control sequence (AAAAAAAA). Phosphorylation of downstream signaling molecules, extracellular signal-regulated protein kinase 1/2 (ERK1/2) and Elk1, was also decreased by DOX-induced miR-15a/16 expression. In addition to MEK1, ERK1 was subsequently found to be targeted by miR-15a/16, with DOX-induced miR-15a/16 reducing total ERK1 levels in T cells. These findings show that TCR stimulation reduces miR-15a/16 levels at early stages of T-cell activation to facilitate increased MEK1 and ERK1, which promotes the sustained MEK1-ERK1/2-Elk1 signaling required for optimal proliferation.
尽管 microRNA（miR）在恶性肿瘤和肿瘤进展方面已经得到广泛研究，但它们在调节 T 细胞活化中的功能尚不清楚。在最初的研究中，我们发现 T 细胞受体（TCR）刺激后 3 至 18 小时 miR-15a/16 的水平降低，表明该 miR 对降低 T 细胞激活的作用。为了进一步探讨这一点，我们开发了一种诱导型 miR15a/16 转基因小鼠模型，以确定在激活的早期阶段升高 miR-15a/16 水平如何影响 T 细胞增殖，并确定该 miR 对 TCR 信号通路的调节。TCR 刺激后 0 至 18 小时用强力霉素（DOX）诱导 miR-15a/16 的表达，降低了 ex vivo T 细胞增殖和体内抗原特异性 T 细胞增殖。我们还结合生物信息学和蛋白质组学方法，鉴定丝裂原激活的蛋白激酶激酶 1（MEK1）（Map2k1）是 miR-15a/16 的靶标。使用 miR 模拟物证实了 miR-15a/16 对 Map2k1 的靶向作用，该模拟物降低了包含 3'-UTR 靶核苷酸序列（UGCUGCUA）的 Map2k1 mRNA，但不降低包含突变对照序列（AAAAAAAA）的 Map2k1。下游信号分子细胞外信号调节蛋白激酶 1/2（ERK1/2）和 Elk1 的磷酸化也因 DOX 诱导的 miR-15a/16 表达而降低。除了 MEK1，ERK1 随后被发现是 miR-15a/16 的靶标，DOX 诱导的 miR-15a/16 降低了 T 细胞中总 ERK1 水平。这些发现表明，TCR 刺激在 T 细胞激活的早期阶段降低 miR-15a/16 水平，以促进 MEK1 和 ERK1 的增加，从而促进持续的 MEK1-ERK1/2-Elk1 信号传导，这是最佳增殖所必需的。
