Golovina Elena, Kokavec Juraj, Kazantsev Dmitry, Yurikova Oxana, Bajecny Martin, Savvulidi Filipp Georgijevic, Simersky Radim, Lenobel Rene, Tost Jorg, Herynek Vit, Sefc Ludek, Sebela Marek, Klener Pavel, Zemanova Zuzana, Stopka Tomas, Vargova Karina Savvulidi
Institute of Pathological Physiology, First Faculty of Medicine, Charles University, Prague, Czech Republic.
Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University, First Faculty of Medicine, Charles University, Vestec, Czech Republic.
Arch Med Res. 2025 Apr;56(3):103124. doi: 10.1016/j.arcmed.2024.103124. Epub 2024 Nov 25.
Cell cycle progression and leukemia development are tightly regulated processes in which even a small imbalance in the expression of cell cycle regulatory molecules and microRNAs (miRNAs) can lead to an increased risk of cancer/leukemia development. Here, we focus on the study of a ubiquitous, multifunctional, and oncogenic miRNA-hsa-miR-155-5p (miR-155, MIR155HG), which is overexpressed in malignancies including chronic lymphocytic leukemia (CLL). Nonetheless, the precise mechanism of how miR-155 regulates the cell cycle in leukemic cells remains the subject of extensive research.
We edited the CLL cell line MEC-1 by CRISPR/Cas9 to introduce a short deletion within the MIR155HG gene. To describe changes at the transcriptome and miRNome level in miR-155-deficient cells, we performed mRNA-seq/miRNA-seq and validated changes by qRT-PCR. Flow cytometry was used to measure cell cycle kinetics. A WST-1 assay, hemocytometer, and Annexin V/PI staining assessed cell viability and proliferation.
The limited but phenotypically robust miR-155 modification impaired cell proliferation, cell cycle, and cell ploidy. This was accompanied by overexpression of the negative cell cycle regulator p21/CDKN1A and Cyclin D1 (CCND1). We confirmed the overexpression of canonical miR-155 targets such as PU.1, FOS, SHIP-1, TP53INP1 and revealed new potential targets (FCRL5, ISG15, and MX1).
We demonstrate that miR-155 deficiency impairs cell proliferation, cell cycle, transcriptome, and miRNome via deregulation of the MIR155HG/TP53INP1/CDKN1A/CCND1 axis. Our CLL model is valuable for further studies to manipulate miRNA levels to revert highly aggressive leukemic cells to nearly benign or non-leukemic types.
细胞周期进程和白血病发展是受到严格调控的过程，其中细胞周期调控分子和微小RNA（miRNA）表达的哪怕是微小失衡都可能导致癌症/白血病发展风险增加。在此，我们聚焦于对一种普遍存在、多功能且具有致癌性的miRNA——hsa-miR-155-5p（miR-155，MIR155HG）的研究，其在包括慢性淋巴细胞白血病（CLL）在内的恶性肿瘤中过表达。尽管如此，miR-155如何调节白血病细胞中的细胞周期的确切机制仍是广泛研究的主题。
我们通过CRISPR/Cas9编辑CLL细胞系MEC-1，以在MIR155HG基因内引入一个短缺失。为了描述miR-155缺陷细胞在转录组和miRnome水平的变化，我们进行了mRNA测序/miRNA测序，并通过qRT-PCR验证变化。流式细胞术用于测量细胞周期动力学。WST-1检测、血细胞计数器和膜联蛋白V/碘化丙啶染色评估细胞活力和增殖。
有限但表型上明显的miR-155修饰损害了细胞增殖、细胞周期和细胞倍性。这伴随着负性细胞周期调节因子p21/CDKN1A和细胞周期蛋白D1（CCND1）的过表达。我们证实了典型miR-155靶标的过表达，如PU.1、FOS、SHIP-1、TP53INP1，并揭示了新的潜在靶标（FCRL5、ISG15和MX1）。
我们证明miR-155缺陷通过MIR155HG/TP53INP1/CDKN1A/CCND1轴的失调损害细胞增殖、细胞周期、转录组和miRnome。我们的CLL模型对于进一步研究操纵miRNA水平以将高度侵袭性的白血病细胞恢复为几乎良性或非白血病类型具有重要价值。
