Page 173 - Haematologica - Vol. 105 n. 6 - June 2020

DARS-AS1 promotes myeloma malignancy in hypoxia
RNA-binding motif protein 39 (RBM39) is a transcrip- tional coactivator for the steroid nuclear receptors ESR1/ER-α and ESR2/ER-β as well as for JUN/AP-1 and NF-κB. RBM39 is also involved in the pre-mRNA splicing process.8-11 Previous studies have pinpointed RBM39 as a proto-oncogene with important roles in the development and progression of numerous types of malignancies.8,12,13 However, the role of RBM39 in MM is largely unknown.
By analysis of high-throughput RNA sequencing results, we identified that lncRNA DARS-AS1 was significantly upregulated in myeloma cells under hypoxic conditions. We evaluated its biological role and clinical significance in tumor progression and revealed that the novel HIF- 1/DARS-AS1/RBM39 signaling pathway is implicated in the tumorigenesis of MM.
Methods
Cell culture and transfection
RPMI 8226, LP-1, U266, H929, and HEK293T cells were obtained from the Cell Resource Center of Shanghai Institute for Biological Science, Chinese Academy of Science. All the cell lines have been authenticated using short tandem repeat geno- type detection. Myeloma cell lines were grown in RPMI 1640 (Gibco, CA, USA) containing 1% penicillin and streptomycin (Fisher Scientific, MA, USA), supplemented with 10% fetal bovine serum (Sigma-Aldrich, MO, USA). The hypoxic environ- ment (1% O2) was generated by flushing a 94% N2/5% CO2 mixture into the incubator. Retroviral particles were produced in HEK293T cells by transient co-transfection of target gene expression vectors, Gag-pol, and VSVG packaging plasmid, while lentiviral particles were produced by transient co-transfec- tion of target gene expression vectors, psPAX2 and pMD2.G packaging plasmid, using lipofectamine 2000 (Invitrogen, MA, USA), according to the manufacturer's instructions. Cells were infected in culture medium supplemented with polybrene (8 μg/mL) and 24 h later the medium was changed to RPMI 1640 with 10% fetal bovine serum. Studies were conducted in accor- dance with the Declaration of Helsinki, and approval was received from the relevant ethics committees.
Luciferase assay
For DARS-AS1 promoter luciferase reporter assays, luciferase reporter constructs (100 ng) containing the potential HIF response element (HRE) sequence [wildtype, (WT) or mutant (MUT)], together with pSV40-renilla (10 ng), and HIF-1α overex- pression plasmids (800 ng) were transfected into HEK293T cells in 24-well plates. Luciferase activity assays were performed 48 h after transfection (Promega, Madison, USA). Firefly luciferase activity was normalized to the corresponding renilla luciferase activity by using the Dual-Luciferase Reporter Assay System. All experiments were performed three times.
Co-immunoprecipitation
Corresponding antibody or IgG was added to cell lysate that was treated with A+G agarose beads (Thermo Fisher Scientific, MA, USA). The mixture was rotated for 6 h and precipitated beads were washed three times with phosphate-buffered saline containing a protease inhibitor cocktail.
RNA immunoprecipitation
RNA immunoprecipitation experiments were performed using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Darmstadt, Germany) according to the manufac-
turer’s instructions. Supernatants were incubated with 1-2 μg of anti-Flag (Sigma, MO, USA) or mouse IgG (Invitrogen, MA, USA) for 6 h at 4°C followed by precipitation with protein A/G agarose (Pierce, Rockforld, IL, USA). The co-precipitated RNA were detected by quantitative real-time reverse transcription polymerase chain reaction (Q-PCR).
RNA pulldown and mass spectrometry analysis
Full-length DARS-AS1 were constructed by subcloning the gene sequences into a pcDNA3.1 (+) backbone. Biotin-labeled DARS-AS1 was synthesized using Biotin RNA Labeling Mix (Roche; Basel, Switzerland) by T7 RNA polymerase (Promega; WI, USA), treated with RNase-free DNase I (Promega) and puri- fied with an RNeasy Mini Kit (QIAGEN). Thirty micrograms of 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, USA) after incubation overnight. The samples were separated by electrophoresis and the specific bands were identified using mass spectrometry and a human proteomic library.
Chromatin immunoprecipitation
HEK293T cells with HIF-1α overexpression (2×107) were har- vested and chromatin immunoprecipitation experiments were performed using a SimpleChIP® Enzymatic Chromatin IP Kit (Agarose Beads) (CST, MA, USA) according to the manufactur- er’s instructions. The primers used are listed in Online Supplementary Table S3.
Bioinformatics analysis
Uniprot (https://www.uniprot.org/) was used to analyze the pro- tein-protein interactions.
Statistics
Continuous variables are expressed as the mean ± standard error (SE). For comparisons of two groups, a t-test was used. When comparing more than two groups, analysis of variance with the Tukey test for multiple comparisons was used. Statistical significance is defined as: *P<0.05, **P<0.01 and ***P<0.001.
Results
DARS-AS1 is upregulated under hypoxia in myeloma cells
To identify hypoxia-regulated lncRNA, we analyzed the difference in the RNA-sequencing data of U266 cells in normoxic and hypoxic culture environments (Online Supplementary Table S4). Twenty-two upregulated lncRNA in U266 cells were successfully identified (Figure 1A). Q-PCR revealed that two lncRNA, DARS-AS1 and MIR210HG, were significantly upregulated under the hypoxic condition (Figure 1B). The modulation of MIR210HG did not have significant effects on myeloma cell proliferation or apoptosis (Online Supplementary Figure S1A-D). Thus, we mainly studied the function of DARS- AS1 in myeloma.
First, we assessed the expression of DARS-AS1 in myeloma cell lines. We found that the hypoxic environ- ment (1% oxygen concentration) caused a significant increase in the level of expression of DARS-AS1 in the MM cell lines (Figure 1C). The expression of DARS-AS1 in CD38+ cells derived from bone marrow of patients with active myeloma was significantly higher than that of nor-
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