Division of Molecular Genome Analysis, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, 69120, Heidelberg, Germany.
Medical Faculty Heidelberg, University of Heidelberg, Im Neuenheimer Feld 672, 69120, Heidelberg, Germany.
J Exp Clin Cancer Res. 2022 Jun 2;41(1):190. doi: 10.1186/s13046-022-02380-8.
MicroRNAs (miRNAs) and isomiRs play important roles in tumorigenesis as essential regulators of gene expression. 5'isomiRs exhibit a shifted seed sequence compared to the canonical miRNA, resulting in different target spectra and thereby extending the phenotypic impact of the respective common pre-miRNA. However, for most miRNAs, expression and function of 5'isomiRs have not been studied in detail yet. Therefore, this study aims to investigate the functions of miRNAs and their 5'isomiRs.
The expression of 5'isomiRs was assessed in The Cancer Genome Atlas (TCGA) breast cancer patient dataset. Phenotypic effects of miR-183 overexpression in triple-negative breast cancer (TNBC) cell lines were investigated in vitro and in vivo by quantifying migration, proliferation, tumor growth and metastasis. Direct targeting of E2F1 by miR-183-5p|+2 was validated with a 3'UTR luciferase assay and linked to the phenotypes of isomiR overexpression.
TCGA breast cancer patient data indicated that three variants of miR-183-5p are highly expressed and upregulated, namely miR-183-5p|0, miR-183-5p|+1 and miR-183-5p|+2. However, TNBC cell lines displayed reduced proliferation and invasion upon overexpression of pre-miR-183. While invasion was reduced individually by all three isomiRs, proliferation and cell cycle progression were specifically inhibited by overexpression of miR-183-5p|+2. Proteomic analysis revealed reduced expression of E2F target genes upon overexpression of this isomiR, which could be attributed to direct targeting of E2F1, specifically by miR-183-5p|+2. Knockdown of E2F1 partially phenocopied the effect of miR-183-5p|+2 overexpression on cell proliferation and cell cycle. Gene set enrichment analysis of TCGA and METABRIC patient data indicated that the activity of E2F strongly correlated with the expression of miR-183-5p, suggesting transcriptional regulation of the miRNA by a factor of the E2F family. Indeed, in vitro, expression of miR-183-5p was regulated by E2F1. Hence, miR-183-5p|+2 directly targeting E2F1 appears to be part of a negative feedback loop potentially fine-tuning its activity.
This study demonstrates that 5'isomiRs originating from the same arm of the same pre-miRNA (i.e. pre-miR-183-5p) may exhibit different functions and thereby collectively contribute to the same phenotype. Here, one of three isomiRs was shown to counteract expression of the pre-miRNA by negatively regulating a transcriptional activator (i.e. E2F1). We speculate that this might be part of a regulatory mechanism to prevent uncontrolled cell proliferation, which is disabled during cancer progression.
微小 RNA（miRNAs）和同型微小 RNA（isomiRs）作为基因表达的重要调控因子，在肿瘤发生中发挥重要作用。5' 同型微小 RNA 与经典 miRNA 相比，其种子序列发生了偏移，导致不同的靶标谱，从而扩展了各自常见前体微小 RNA 的表型影响。然而，对于大多数 miRNA 来说，其 5' 同型微小 RNA 的表达和功能尚未得到详细研究。因此，本研究旨在探讨 miRNA 及其 5' 同型微小 RNA 的功能。
在癌症基因组图谱（TCGA）乳腺癌患者数据集评估 5' 同型微小 RNA 的表达。通过定量迁移、增殖、肿瘤生长和转移，在三阴性乳腺癌（TNBC）细胞系中研究 miR-183 过表达的表型效应。通过 3'UTR 荧光素酶测定验证 miR-183-5p|+2 对 E2F1 的直接靶向作用，并将其与同型微小 RNA 过表达的表型联系起来。
TCGA 乳腺癌患者数据表明，三种 miR-183-5p 的变体高度表达且上调，即 miR-183-5p|0、miR-183-5p|+1 和 miR-183-5p|+2。然而，TNBC 细胞系在过表达前体微小 RNA 时表现出增殖和侵袭能力降低。虽然所有三种同型微小 RNA 都能单独降低侵袭能力，但增殖和细胞周期进程仅被 miR-183-5p|+2 的过表达特异性抑制。蛋白质组学分析表明，该同型微小 RNA 过表达后 E2F 靶基因的表达减少，这归因于 E2F1 的直接靶向作用，特别是 miR-183-5p|+2。E2F1 的敲低部分模拟了 miR-183-5p|+2 过表达对细胞增殖和细胞周期的影响。TCGA 和 METABRIC 患者数据的基因集富集分析表明，E2F 的活性与 miR-183-5p 的表达强烈相关，提示 E2F 家族的一个因子对 miRNA 的转录调控。事实上，在体外，miR-183-5p 的表达受到 E2F1 的调控。因此，miR-183-5p|+2 对 E2F1 的直接靶向作用似乎是其活性的负反馈回路的一部分，可能有助于精细调节其活性。
本研究表明，源自同一前体微小 RNA（即 pre-miR-183-5p）同一臂的 5' 同型微小 RNA 可能具有不同的功能，从而共同促成相同的表型。在这里，三种同型微小 RNA 之一被证明通过负向调节转录激活因子（即 E2F1）来抵消前体微小 RNA 的表达。我们推测，这可能是一种防止失控性细胞增殖的调控机制的一部分，而这种机制在癌症进展过程中会被禁用。
