ARTICLE Plasma Cell Disorders Hypoxia-induced long non-coding RNA Ferrata Storti Foundation DARS-AS1 regulates RBM39 stability to promote myeloma malignancy Jia Tong,1,* Xiaoguang Xu,1,* Zilu Zhang,1 Chengning Ma,2 Rufang Xiang,1 Jia Liu,1 Wenbin Xu,1 Chao Wu,1 Junmin Li,1 Fenghuang Zhan,3 Yingli Wu2 and Hua Yan1,4 1Department of Hematology, Affiliated Ruijin Hospital of Shanghai Jiao Tong University Haematologica 2020 School of Medicine, Shanghai, China; 2Hongqiao International Institute of Medicine, Volume 105(6):1630-1640 Shanghai Tongren Hospital, Key Laboratory of Cell Differentiation and Apoptosis of the Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai, China; 3Division of Hematology, Oncology, and Blood and Marrow Transplantation, Department of Internal Medicine, University of Iowa, Iowa City, IA, USA and 4Department of General Practice, Ruijin Hospital, Shanghai Jiao Tong Un iversity School of Medicine, Shanghai, China. *JA and XX contributed equally as co-first authors. ABSTRACT ultiple myeloma is a malignant plasma-cell disease, which is highly dependent on the hypoxic bone marrow microenvironment. MHowever, the underlying mechanisms of hypoxia contributing to myeloma genesis are not fully understood. Here, we show that long non- coding RNA DARS-AS1 in myeloma is directly upregulated by hypoxia inducible factor (HIF)-1. Importantly, DARS-AS1 is required for the survival Correspondence: and tumorigenesis of myeloma cells both in vitro and in vivo. DARS-AS1 HUA YAN exerts its function by binding RNA-binding motif protein 39 (RBM39), [email protected] which impedes the interaction between RBM39 and its E3 ubiquitin ligase RNF147, and prevents RBM39 from degradation. The overexpression of YINGLI WU RBM39 observed in myeloma cells is associated with poor prognosis. [email protected] Furthermore, knockdown of DARS-AS1 inhibits the mammalian target of rapamycin signaling pathway, an effect that is reversed by RBM39 overex- Received: February 1, 2019. pression. We reveal that a novel HIF-1/DARS-AS1/RBM39 pathway is impli- Accepted: July 5, 2019. cated in the pathogenesis of myeloma. Targeting DARS-AS1/RBM39 may, therefore, represent a novel strategy to combat myeloma. Pre-published: July 9, 2019. doi:10.3324/haematol.2019.218289 Introduction Check the online version for the most updated Multiple myeloma (MM) is characterized by abnormal accumulation of mono- information on this article, online supplements, clonal plasma cells in the bone marrow and broad clinical and pathophysiological and information on authorship & disclosures: heterogeneity leading to a fatal outcome. Hypoxia in specific bone marrow niches www.haematologica.org/content/105/6/1630 induces numerous changes in the expression of genes that contribute to the main- tenance of myeloma cells and the progression of myeloma, leading to drug resist- ance and cancer recurrence.1 Focusing on the hypoxia-related pathogenic mecha- ©2020 Ferrata Storti Foundation nisms in myeloma may facilitate the optimization of the choice of therapeutic reg- Material published in Haematologica is covered by copyright. imen and improve clinical outcomes. All rights are reserved to the Ferrata Storti Foundation. Use of Long non-coding RNA (lncRNA) are mRNA-like transcripts that are longer than published material is allowed under the following terms and 2,3 conditions: 200 nucleotides. Accumulating evidence indicates that lncRNA are crucial mole- https://creativecommons.org/licenses/by-nc/4.0/legalcode. cules that participate in gene regulation at the epigenetic, transcriptional, and post- Copies of published material are allowed for personal or inter- transcriptional levels.3,4 Aberrant expression of lncRNA can promote the development nal use. Sharing published material for non-commercial pur- and progression of malignant tumors through contributing to proliferation, invasion poses is subject to the following conditions: 5-7 https://creativecommons.org/licenses/by-nc/4.0/legalcode, and metastasis. lncRNA may, therefoe, serve as potential diagnostic biomarkers and sect. 3. Reproducing and sharing published material for com- therapeutic targets for cancers. However, few lncRNA have been functionally studied mercial purposes is not allowed without permission in writing in MM. Many hypoxia inducible factor (HIF)-dependent protein-coding genes con- from the publisher. tribute to adaptation to hypoxia. Whether lncRNA are involved in the response to hypoxia in myeloma and the determination of their regulatory roles is important for a better understanding of the development and progression of myeloma. 1630 haematologica | 2020; 105(6) DARS-AS1 promotes myeloma malignancy in hypoxia RNA-binding motif protein 39 (RBM39) is a transcrip- turer’s instructions. Supernatants were incubated with 1-2 mg of tional coactivator for the steroid nuclear receptors anti-Flag (Sigma, MO, USA) or mouse IgG (Invitrogen, MA, ESR1/ER-α and ESR2/ER-β as well as for JUN/AP-1 and USA) for 6 h at 4°C followed by precipitation with protein A/G NF-κB. RBM39 is also involved in the pre-mRNA splicing agarose (Pierce, Rockforld, IL, USA). The co-precipitated RNA process.8-11 Previous studies have pinpointed RBM39 as a were detected by quantitative real-time reverse transcription proto-oncogene with important roles in the development polymerase chain reaction (Q-PCR). and progression of numerous types of malignancies.8,12,13 However, the role of RBM39 in MM is largely unknown. RNA pulldown and mass spectrometry analysis By analysis of high-throughput RNA sequencing results, Full-length DARS-AS1 were constructed by subcloning the we identified that lncRNA DARS-AS1 was significantly gene sequences into a pcDNA3.1 (+) backbone. Biotin-labeled upregulated in myeloma cells under hypoxic conditions. DARS-AS1 was synthesized using Biotin RNA Labeling Mix We evaluated its biological role and clinical significance in (Roche; Basel, Switzerland) by T7 RNA polymerase (Promega; tumor progression and revealed that the novel HIF- WI, USA), treated with RNase-free DNase I (Promega) and puri- 1/DARS-AS1/RBM39 signaling pathway is implicated in fied with an RNeasy Mini Kit (QIAGEN). Thirty micrograms of the tumorigenesis of MM. biotinylated RNA were used in each pulldown assay. The pro- teins with biotin-labeled DARS-AS1 were pulled down with streptavidin magnetic beads (Thermo, Fisher SCientific, MA, Methods USA) after incubation overnight. The samples were separated by electrophoresis and the specific bands were identified using Cell culture and transfection mass spectrometry and a human proteomic library. RPMI 8226, LP-1, U266, H929, and HEK293T cells were obtained from the Cell Resource Center of Shanghai Institute for Chromatin immunoprecipitation 7 Biological Science, Chinese Academy of Science. All the cell HEK293T cells with HIF-1α overexpression (2×10 ) were har- lines have been authenticated using short tandem repeat geno- vested and chromatin immunoprecipitation experiments were type detection. Myeloma cell lines were grown in RPMI 1640 performed using a SimpleChIP® Enzymatic Chromatin IP Kit (Gibco, CA, USA) containing 1% penicillin and streptomycin (Agarose Beads) (CST, MA, USA) according to the manufactur- (Fisher Scientific, MA, USA), supplemented with 10% fetal er’s instructions. The primers used are listed in Online bovine serum (Sigma-Aldrich, MO, USA). The hypoxic environ- Supplementary Table S3. ment (1% O2) was generated by flushing a 94% N2/5% CO2 mixture into the incubator. Retroviral particles were produced in Bioinformatics analysis HEK293T cells by transient co-transfection of target gene Uniprot (https://www.uniprot.org/) was used to analyze the pro- expression vectors, Gag-pol, and VSVG packaging plasmid, tein-protein interactions. while lentiviral particles were produced by transient co-transfec- tion of target gene expression vectors, psPAX2 and pMD2.G Statistics packaging plasmid, using lipofectamine 2000 (Invitrogen, MA, Continuous variables are expressed as the mean ± standard USA), according to the manufacturer's instructions. Cells were error (SE). For comparisons of two groups, a t-test was used. infected in culture medium supplemented with polybrene (8 When comparing more than two groups, analysis of variance mg/mL) and 24 h later the medium was changed to RPMI 1640 with the Tukey test for multiple comparisons was used. with 10% fetal bovine serum. Studies were conducted in accor- Statistical significance is defined as: *P<0.05, **P<0.01 and dance with the Declaration of Helsinki, and approval was ***P<0.001. received from the relevant ethics committees. Luciferase assay Results For DARS-AS1 promoter luciferase reporter assays, luciferase reporter constructs (100 ng) containing the potential HIF DARS-AS1 is upregulated under hypoxia in myeloma response element (HRE) sequence [wildtype, (WT) or mutant cells (MUT)], together with pSV40-renilla (10 ng), and HIF-1α overex- To identify hypoxia-regulated lncRNA, we analyzed pression plasmids (800 ng) were transfected into HEK293T cells the difference in the RNA-sequencing data of U266 cells in 24-well plates. Luciferase activity assays were performed 48 h in normoxic and hypoxic culture environments (Online after transfection (Promega, Madison, USA). Firefly luciferase Supplementary Table S4). Twenty-two upregulated activity was normalized to the corresponding renilla luciferase lncRNA in U266 cells were successfully identified (Figure activity by using the Dual-Luciferase Reporter Assay System. All 1A). Q-PCR