Dnmt3a is downregulated by Stat5a and mediates G0/G1 arrest by suppressing the miR-17-5p/Cdkn1a axis in Jak2V617F cells

BMC Cancer - 2021
Jie Zhou1,2, Cheng Guo2,1, Hao Wu3,2, Bing Li3,2, Lili Zhou3,2, Aibin Liang2,3, Jianfei Fu3,2
1Department of Gastroenterology, Tongji Hospital of Tongji University, Shanghai, China
2Tongji University School of Medicine, Shanghai, China
3Department of Hematology, Tongji Hospital of Tongji University, Tongji University School of Medicine, Shanghai, China

Tóm tắt

Abstract Background Despite of the frequently reported Dnmt3a abormality in classical myeloproliferative neoplasms (cMPNs) patients, few research explores how the Dnmt3a is regulated by Jak2V617F mutation. In this study, we have investigated how the Dnmt3a is regulated by Jak2V617F mutation and its effects on downstream signaling pathways in cMPNs. Methods Specimens of Jak2V617F positive cMPN patients and normal controls were collected. Murine BaF3 cell line was used to construct cell models. Dual-Glo luciferase assays and chromatin immunoprecipitation (ChIP)-qPCR were performed to detect the impact of Stat5a on transcription activity of Dnmt3a. Soft agar colony formation assay and cell counting assay were performed to detect cell proliferation. BrdU staining and flow cytometry were used to investigate cell cycle distribution. Western blotting and quantitative reverse-transcription PCR (qPCR) were performed to detect the expression levels of genes. Results Firstly, the results of western blotting and qPCR revealed that compared with the control samples, Dnmt3a is downregulated in Jak2V617F positive samples. Then we explored the mechanism behind it and found that Dnmt3a is a downstream target of Stat5a, the transcription and translation of Dnmt3a is suppressed by the binding of aberrantly activated Stat5a with Dnmt3a promoter in Jak2V617F positive samples. We further revealed the region approximately 800 bp upstream of the first exon of the Dnmt3a promoter, which includes a gamma-activated sequence (GAS) motif of Stat5a, is the specific site that Stat5a binds to. Soft agar colony formation assay, cell counting assay, and BrdU staining and flow cytometry assay found that Dnmt3a in Jak2V617F-BaF3 cells significantly affected the cell proliferation capacity and cell cycle distribution by suppressing Cdkn1a via miR-17-5p/Cdkn1a axis and mediated G0/G1 arrest. Conclusions Transcription and translation of Dnmt3a is downregulated by the binding of Stat5a with Dnmt3a promoter in Jak2V617F cells. The GAS motif at promoter of Dnmt3a is the exact site where the Stat5a binds to. Dnmt3a conducted G0/G1 arrest through regulating miR-17-5p/Cdkn1a axis. The axis of Stat5a/Dnmt3a/miR-17-5p/Cdkn1a potentially provides a treatment target for cMPNs.

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Tài liệu tham khảo

Ozono Y, Shide K, Kameda T, Kamiunten A, Tahira Y, Sekine M, et al. Neoplastic fibrocytes play an essential role in bone marrow fibrosis in Jak2V617F-induced primary myelofibrosis mice. Leukemia. 2021;35(2):454–67. https://doi.org/10.1038/s41375-020-0880-3.

Lundberg P, Karow A, Nienhold R, Looser R, Hao-Shen H, Nissen I, et al. Clonal evolution and clinical correlates of somatic mutations in myeloproliferative neoplasms. Blood. 2014;123(14):2220–8. https://doi.org/10.1182/blood-2013-11-537167.

Vainchenker W, Kralovics R. Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms. Blood. 2017;129(6):667–79. https://doi.org/10.1182/blood-2016-10-695940.

Ginno PA, Gaidatzis D, Feldmann A, Hoerner L, Imanci D, Burger L, et al. A genome-scale map of DNA methylation turnover identifies site-specific dependencies of DNMT and TET activity. Nat Commun. 2020;11(1):2680. https://doi.org/10.1038/s41467-020-16354-x.

Bullinger L, Döhner K, Döhner H. Genomics of acute myeloid leukemia diagnosis and pathways. J Clin Oncol. 2017;35(9):934–46. https://doi.org/10.1200/jco.2016.71.2208.

Jacquelin S, Straube J, Cooper L, Vu T, Song A, Bywater M, et al. Jak2V617F and Dnmt3a loss cooperate to induce myelofibrosis through activated enhancer-driven inflammation. Blood. 2018;132(26):2707–21. https://doi.org/10.1182/blood-2018-04-846220.

Takeuchi A, Nishioka C, Ikezoe T, Yang J, Yokoyama A. STAT5A regulates DNMT3A in CD34(+)/CD38(−) AML cells. Leuk Res. 2015;39(8):897–905. https://doi.org/10.1016/j.leukres.2015.05.006.

Arber DA, Orazi A, Hasserjian R, Thiele J, Borowitz MJ, Le Beau MM, et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood. 2016;127(20):2391–405. https://doi.org/10.1182/blood-2016-03-643544.

Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods. 2001;25(4):402–8. https://doi.org/10.1006/meth.2001.1262.

Soldaini E, John S, Moro S, Bollenbacher J, Schindler U, Leonard WJ. DNA binding site selection of dimeric and tetrameric Stat5 proteins reveals a large repertoire of divergent tetrameric Stat5a binding sites. Mol Cell Biol. 2000;20(1):389–401. https://doi.org/10.1128/mcb.20.1.389-401.2000.

Dutto I, Tillhon M, Cazzalini O, Stivala LA, Prosperi E. Biology of the cell cycle inhibitor p21(CDKN1A): molecular mechanisms and relevance in chemical toxicology. Arch Toxicol. 2015;89(2):155–78. https://doi.org/10.1007/s00204-014-1430-4.

Yang ZG, Awan FM, Du WW, Zeng Y, Lyu J, Wu D, et al. The circular RNA interacts with STAT3, increasing its nuclear translocation and wound repair by modulating Dnmt3a and miR-17 function. Mol Ther. 2017;25(9):2062–74. https://doi.org/10.1016/j.ymthe.2017.05.022.

Flavahan WA, Gaskell E, Bernstein BE. Epigenetic plasticity and the hallmarks of cancer. Science. 2017;357(6348). https://doi.org/10.1126/science.aal2380.

Yang L, Rau R, Goodell MA. DNMT3A in haematological malignancies. Nat Rev Cancer. 2015;15(3):152–65. https://doi.org/10.1038/nrc3895.

Venugopal K, Feng Y, Shabashvili D, Guryanova OA. Alterations to DNMT3A in hematologic malignancies. Cancer Res. 2021;81(2):254–63. https://doi.org/10.1158/0008-5472.can-20-3033.

Chaudry SF, Chevassut TJ. Epigenetic Guardian: a review of the DNA methyltransferase DNMT3A in acute myeloid Leukaemia and clonal Haematopoiesis. Biomed Res Int. 2017;2017:1–13. https://doi.org/10.1155/2017/5473197.

Baumeister J, Chatain N, Hubrich A, Maié T, Costa IG, Denecke B, et al. Hypoxia-inducible factor 1 (HIF-1) is a new therapeutic target in JAK2V617F-positive myeloproliferative neoplasms. Leukemia. 2020;34(4):1062–74. https://doi.org/10.1038/s41375-019-0629-z.

Bartalucci N, Tozzi L, Bogani C, Martinelli S, Rotunno G, Villeval JL, et al. Co-targeting the PI3K/mTOR and JAK2 signalling pathways produces synergistic activity against myeloproliferative neoplasms. J Cell Mol Med. 2013;17(11):1385–96. https://doi.org/10.1111/jcmm.12162.

Rivas ML, Cobreros L, Zeidler MP, Hombría JC. Plasticity of Drosophila Stat DNA binding shows an evolutionary basis for Stat transcription factor preferences. EMBO Rep. 2008;9(11):1114–20. https://doi.org/10.1038/embor.2008.170.

Hayashi M, Futawaka K, Koyama R, Fan Y, Matsushita M, Hirao A, et al. Effects of growth hormone on uncoupling protein 1 in white adipose tissues in obese mice. Growth Hormon IGF Res. 2017;37:31–9. https://doi.org/10.1016/j.ghir.2017.10.006.

Flisikowski K, Szymanowska M, Zwierzchowski L. The DNA-binding capacity of genetic variants of the bovine STAT5A transcription factor. Cell Mol Biol Lett. 2003;8(3):831–40.

Jeong M, Park HJ, Celik H, Ostrander EL, Reyes JM, Guzman A, et al. Loss of Dnmt3a immortalizes hematopoietic stem cells in vivo. Cell Rep. 2018;23(1):1–10. https://doi.org/10.1016/j.celrep.2018.03.025.

Yang L, Rodriguez B, Mayle A, Park HJ, Lin X, Luo M, et al. DNMT3A loss drives enhancer Hypomethylation in FLT3-ITD-associated Leukemias. Cancer Cell. 2016;29(6):922–34. https://doi.org/10.1016/j.ccell.2016.05.003.

Zhang ZM, Lu R, Wang P, Yu Y, Chen D, Gao L, et al. Structural basis for DNMT3A-mediated de novo DNA methylation. Nature. 2018;554(7692):387–91. https://doi.org/10.1038/nature25477.

Mao Q, Wu S, Gu X, Du S, Mo K, Sun L, et al. DNMT3a-triggered downregulation of K (2p) 1.1 gene in primary sensory neurons contributes to paclitaxel-induced neuropathic pain. Int J Cancer. 2019;145(8):2122–34. https://doi.org/10.1002/ijc.32155.

Kataoka I, Funata S, Nagahama K, Isogaya K, Takeuchi H, Abe N, et al. DNMT3A overexpression is associated with aggressive behavior and enteroblastic differentiation of gastric adenocarcinoma. Ann Diagn Pathol. 2020;44:151456. https://doi.org/10.1016/j.anndiagpath.2019.151456.

Haney SL, Upchurch GM, Opavska J, Klinkebiel D, Hlady RA, Roy S, et al. Dnmt3a is a Haploinsufficient tumor suppressor in CD8+ peripheral T cell lymphoma. PLoS Genet. 2016;12(9):e1006334. https://doi.org/10.1371/journal.pgen.1006334.

Qi R, Wang J, Jiang Y, Qiu Y, Xu M, Rong R, et al. Snai1-induced partial epithelial-mesenchymal transition orchestrates p53-p21-mediated G2/M arrest in the progression of renal fibrosis via NF-κB-mediated inflammation. Cell Death Dis. 2021;12(1):44. https://doi.org/10.1038/s41419-020-03322-y.

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