miR-543 regulates the epigenetic landscape of myelofibrosis by targeting TET1 and TET2
Enrique Fuentes-Mattei, … , George A. Calin, Roxana S. Redis
Enrique Fuentes-Mattei, … , George A. Calin, Roxana S. Redis
Published January 16, 2020
Citation Information: JCI Insight. 2020;5(1):e121781. https://doi.org/10.1172/jci.insight.121781.
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Research Article Hematology Oncology

miR-543 regulates the epigenetic landscape of myelofibrosis by targeting TET1 and TET2

Abstract

Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by cytopenia and extramedullary hematopoiesis, resulting in splenomegaly. Multiple pathological mechanisms (e.g., circulating cytokines and genetic alterations, such as JAKV617F mutation) have been implicated in the etiology of MF, but the molecular mechanism causing resistance to JAK2V617F inhibitor therapy remains unknown. Among MF patients who were treated with the JAK inhibitor ruxolitinib, we compared noncoding RNA profiles of ruxolitinib therapy responders versus nonresponders and found miR-543 was significantly upregulated in nonresponders. We validated these findings by reverse transcription–quantitative PCR. in this same cohort, in 2 additional independent MF patient cohorts from the United States and Romania, and in a JAK2V617F mouse model of MF. Both in vitro and in vivo models were used to determine the underlying molecular mechanism of miR-543 in MF. Here, we demonstrate that miR-543 targets the dioxygenases ten-eleven translocation 1 (TET1) and 2 (TET2) in patients and in vitro, causing increased levels of global 5-methylcytosine, while decreasing the acetylation of histone 3, STAT3, and tumor protein p53. Mechanistically, we found that activation of STAT3 by JAKs epigenetically controls miR-543 expression via binding the promoter region of miR-543. Furthermore, miR-543 upregulation promotes the expression of genes related to drug metabolism, including CYP3A4, which is involved in ruxolitinib metabolism. Our findings suggest miR-543 as a potentially novel biomarker for the prognosis of MF patients with a high risk of treatment resistance and as a potentially new target for the development of new treatment options.

Authors

Enrique Fuentes-Mattei, Recep Bayraktar, Taghi Manshouri, Andreia M. Silva, Cristina Ivan, Diana Gulei, Linda Fabris, Nayra Soares do Amaral, Pilar Mur, Cristina Perez, Elizabeth Torres-Claudio, Mihnea P. Dragomir, Adriana Badillo-Perez, Erik Knutsen, Pranav Narayanan, Leonard Golfman, Masayoshi Shimizu, Xinna Zhang, Wanke Zhao, Wanting Tina Ho, Marcos Roberto Estecio, Geoffrey Bartholomeusz, Ciprian Tomuleasa, Ioana Berindan-Neagoe, Patrick A. Zweidler-McKay, Zeev Estrov, Zhizhuang J. Zhao, Srdan Verstovsek, George A. Calin, Roxana S. Redis

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Figure 5

TET1 and TET2 are direct targets of miR-543.

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TET1 and TET2 are direct targets of miR-543. (A) Top: Western blot analy...
(A) Top: Western blot analysis of TET1 and TET2 proteins in BM samples from patients with MF positive for the JAK2V617F mutation who responded to ruxolitinib treatment (complete elimination of palpable splenomegaly, low miR-543) or did not respond to ruxolitinib treatment (0%–24% spleen size reduction, high miR-543). GAPDH (for TET1) or β-actin (for TET2) protein levels were used as normalizers. Bottom: Expression levels of miR-543 in the peripheral blood (PB) samples derived from the same patients. Relative expression of miR-543 was measured by RT-qPCR and normalized to U6 and RNU48 levels. (B) Representative Western blot analysis of TET1 and TET2 proteins in K562 cells stably transduced with empty vector (control) or p–miR-543 expression vector. GAPDH or β-actin protein levels were used as normalizers. Relative expression of miR-543 measured by RT-qPCR and normalized to U6 and RNU48 levels (n = 3; bottom). (C) Representative Western blot analysis of TET1 and TET2 proteins in OCI-AML3 cells stably transduced with empty vector (control) or p–miR-543 expression vector. Vinculin or β-actin protein levels were used as normalizers. Relative expression of miR-543 measured by RT-qPCR and normalized to U6 and RNU48 levels (n = 3; bottom). (D) Expression of TET1 or TET2 in K562 cells stably transduced with STAT3 shRNA or control shRNA treated with ruxolitinib after being transiently cotransfected with the miR-543 mimic or control miRNA (scramble mimic). Results presented as relative mRNA levels relative to the geometric mean of the 2 normalizers: β-actin and GAPDH. (E) Levels of TET1 and TET2 mRNAs bound to the Ago2 complex were pulled down with IgG or anti-Ago2 antibody from HEK293 cells transiently transfected with empty vector (control) or with p–miR-543 expression vector. Two-tailed t test was used to assess statistical significance for B and C. Mann-Whitney-Wilcoxon nonparametric test was used for D. All experiments were performed independently 2 or 3 times, and representative blots are shown. Ago2, protein argonaute-2; RIP, RNA immunoprecipitation; NS, not significant.