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miR-24-3p Is Overexpressed in Hodgkin Lymphoma and Protects Hodgkin and Reed-Sternberg Cells from Apoptosis

Open ArchivePublished:April 19, 2017DOI:https://doi.org/10.1016/j.ajpath.2017.02.016
      miRNAs play important roles in biological processes, such as proliferation, metabolism, differentiation, and apoptosis, whereas altered expression levels contribute to diseases, such as cancers. We identified miRNAs with aberrant expression in Hodgkin lymphoma (HL) and investigated their role in pathogenesis. Small RNA sequencing revealed 84 significantly differentially expressed miRNAs in HL cell lines as compared to germinal center B cells. Three up-regulated miRNAs—miR-23a-3p, miR-24-3p, and miR-27a-3p—were derived from one primary miRNA transcript. Loss-of-function analyses for these miRNAs and their seed family members resulted in decreased growth on miR-24-3p inhibition in three HL cell lines and of miR-27a/b-3p inhibition in one HL cell line. Apoptosis analysis indicated that the effect of miR-24-3p on cell growth is at least in part caused by an increase of apoptotic cells. Argonaute 2 immunoprecipitation revealed 1142 genes consistently targeted by miRNAs in at least three of four HL cell lines. Furthermore, 52 of the 1142 genes were predicted targets of miR-24-3p. Functional annotation analysis revealed a function related to cell growth, cell death, and/or apoptosis for 15 of the 52 genes. Western blotting of the top five genes showed increased protein levels on miR-24-3p inhibition for CDKN1B/P27kip1 and MYC. In summary, we showed that miR-24-3p is up-regulated in HL and its inhibition impairs cell growth possibly via targeting CDKN1B/P27kip1 and MYC.
      Hodgkin lymphoma (HL) is a B-cell–derived lymphoma classified into classic HL (cHL) and nodular lymphocyte –predominant HL.
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      The World Health Organization (WHO) classification of tumors of the hematopoietic and lymphoid tissues: an overview with emphasis on the myeloid neoplasms.
      Nodular lymphocyte–predominant HL is a more rare subtype of HL, accounting for approximately 5% of all cases.
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      • LaCasce A.S.
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      Hodgkin and Reed-Sternberg cells in Hodgkin's disease represent the outgrowth of a dominant tumor clone derived from (crippled) germinal center B cells.
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      One single miRNA can interact with multiple targets.
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      MicroRNAs: target recognition and regulatory functions.
      The first human cancer type reported to be associated with miRNAs was chronic lymphocytic leukemia.
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      MicroRNAs as oncogenes.
      So far, multiple miRNAs are deregulated in B-cell lymphoma and for a subset of them pivotal functions have been shown in the pathogenesis.
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      • Negrini M.
      • Bullrich F.
      • Croce C.M.
      Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia.
      • van den Berg A.
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      • Maggio E.
      • Poppema S.
      High expression of B-cell receptor inducible gene BIC in all subtypes of Hodgkin lymphoma.
      • Medina P.P.
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      • Slack F.J.
      OncomiR addiction in an in vivo model of microRNA-21-induced pre-B-cell lymphoma.
      Using small RNA sequencing, Landgraf et al
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      • Rusu M.
      • Sheridan R.
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      • Iovino N.
      • Aravin A.
      • et al.
      A mammalian microRNA expression atlas based on small RNA library sequencing.
      generated among others miRNA expression profiles of four Epstein-Barr virus–negative cHL cell lines. Van Vlierberghe et al
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      • Van Roy N.
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      • Poppe B.
      • Speleman F.
      Comparison of miRNA profiles of microdissected Hodgkin/Reed-Sternberg cells and Hodgkin cell lines versus CD77+ B-cells reveals a distinct subset of differentially expressed miRNAs.
      identified 12 up-regulated and three down-regulated miRNAs in microdissected HRS cells from nine cHL patients and HL cell lines compared to CD77+ germinal center (GC)-B cells. Gibcus et al
      • Gibcus J.H.
      • Tan L.P.
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      • de Jong D.
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      • Moller P.
      • Poppema S.
      • Kroesen B.J.
      • van den Berg A.
      Hodgkin lymphoma cell lines are characterized by a specific miRNA expression profile.
      determined the miRNA profile of HL cell lines in comparison to GC-B cell–derived lymphoblastoid cell lines and other B-cell lymphoma cell lines and showed increased expression of the miR-17∼92 cluster, miR-16, miR-21, miR-24, and miR-155 in HL. Functional studies in HL are limited, but for some of the miRNAs their putative role has been established. miR-135a targets JAK2, which leads to reduced Bcl-xL levels in HL.
      • Navarro A.
      • Diaz T.
      • Martinez A.
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      • Ferrer G.
      • Martinez C.
      • Montserrat E.
      • Monzo M.
      Regulation of JAK2 by miR-135a: prognostic impact in classic Hodgkin lymphoma.
      The miR-17/106b seed family targets CDKN1A encoding for the P21 protein and inhibition of this seed family results in a G1-phase cell cycle arrest.
      • Gibcus J.H.
      • Kroesen B.J.
      • Koster R.
      • Halsema N.
      • de Jong D.
      • de Jong S.
      • Poppema S.
      • Kluiver J.
      • Diepstra A.
      • van den Berg A.
      MiR-17/106b seed family regulates p21 in Hodgkin's lymphoma.
      HuR and Dicer were shown to be targets of the oncogenic miR-9 and inhibition of miR-9 resulted in higher cytokine production levels.
      • Leucci E.
      • Zriwil A.
      • Gregersen L.H.
      • Jensen K.T.
      • Obad S.
      • Bellan C.
      • Leoncini L.
      • Kauppinen S.
      • Lund A.H.
      Inhibition of miR-9 de-represses HuR and DICER1 and impairs Hodgkin lymphoma tumour outgrowth in vivo.
      A significant correlation between miR-124a methylation status and a high-risk international prognostic score was found in HL.
      • Ben Dhiab M.
      • Ziadi S.
      • Ksiaa F.
      • Louhichi T.
      • Ben Gacem R.
      • Ben Zineb A.
      • Amara K.
      • Hachana M.
      • Trimeche M.
      Methylation of miR124a-1, miR124a-2, and miR124a-3 in Hodgkin lymphoma.
      Herein, we established an HL-specific miRNA expression profile using small RNA sequencing and validated differential expression of selected miRNAs. Furthermore, we determined the effects of miR-23a/b-3p, miR-24-3p, and miR-27a/b-3p inhibition on cell growth. To identify target genes regulated by these miRNAs, Ago2 RNA immunoprecipitation (Ago2-RIP) followed by a microarray analysis was performed on four HL cell lines. Targeting of selected Ago2-IP–enriched miR-24-3p–target genes was confirmed using Western blotting.

      Materials and Methods

      Culturing of HL Cell Lines and Sorting of GC B Cells

      L540 (nodular sclerosis, T-cell derived), KM-H2 (mixed cellularity), L1236 (mixed cellularity), L428 (nodular sclerosis), and HDLM2 (nodular sclerosis) HL cell lines were cultured in RPMI 1640 medium (Cambrex Biosciences, Walkersville, MD), and the SUPHD1 (lymphocyte depleted) HL cell line was cultured in McCoy 5A medium (Cambrex Biosciences) at 37°C in an atmosphere containing 5% CO2. Culture medium was supplemented with 2 mmol/L ultraglutamine 1 (Cambrex Biosciences), 100 U/mL penicillin/streptomycin, and 5% L428, 10% L1236, KM-H2, and HDLM2, or 20% L540 and SUPHD1 fetal bovine serum (Cambrex Biosciences).
      GC-B cells were sorted from tonsil tissue samples of three donors aged between 2 and 6 years. Two of the three GC-B cells were purified >98% from human tonsils based on expression of CD20+lgDCD38+ as previously described.
      • Robertus J.L.
      • Kluiver J.
      • Weggemans C.
      • Harms G.
      • Reijmers R.M.
      • Swart Y.
      • Kok K.
      • Rosati S.
      • Schuuring E.
      • van Imhoff G.
      • Pals S.T.
      • Kluin P.
      • van den Berg A.
      MiRNA profiling in B non-Hodgkin lymphoma: a MYC-related miRNA profile characterizes Burkitt lymphoma.
      The third sample was magnetic-activated cell sorting purified >95% based on expression of IgDCD138CD3CD10+. Briefly, a freshly prepared tonsillar cell suspension was prepared and depleted from IgD+ (naïve), CD138+ (plasma cells), and CD3+ (T cells) using LD columns and IgDBiotin+ anti-Biotin beads, CD138-beads, and CD3-beads (Miltenyi Biotec, Leiden, the Netherlands). Next, we positively enriched the flow-through fraction for CD10+ cells using CD10 beads and LS columns (Miltenyi Biotec). Purity of the GC-B cell population was confirmed by fluorescence-activated cell sorting using antibodies against CD20, IgD, and CD38 as indicated above. All cells of CD20+IgDCD38+ were considered to be GC-B cells. The procedures were according to the guidelines of the medical ethics board of the University Medical Center Groningen. Written informed consent was obtained for the use of the tonsil samples from the parents of the children.

      RNA Isolation

      RNA was isolated from the total cell lysate fractions and the Ago2-IP fractions of HL cells using miRNeasy mini kit (Qiagen, Hiden, Germany), according to manufacturer's protocol. The RNA concentration was measured by a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA), and the integrity was evaluated on a 1% agarose gel.

      Small RNA Library Preparation, Sequencing, and Data Analysis

      Small RNA libraries were generated from 2000 ng total RNA using TruSeq Small RNA Sample Preparation Kit and TruSeq small RNA indices (Illumina, San Diego, CA). All RNA samples were analyzed on an Illumina 2000 HiSeq high-throughput sequencing platform. Briefly, 3′- and 5′-adaptor sequences were removed using the CLC Genomics Workbench (CLC Bio, Cambridge, MA). RNA reads were analyzed with miRDeep version 2.0 (Max Delbrück Center for Molecular Medicine in the Helmholtz Association, https://www.mdc-berlin.de/8551903/en)
      • Friedländer M.R.
      • Chen W.
      • Adamidi C.
      • Maaskola J.
      • Einspanier R.
      • Knespel S.
      • Rajewsky N.
      Discovering microRNAs from deep sequencing data using miRDeep.
      and annotated against miRBASE version 21 (http://www.mirbase.org, last accessed November 11, 2014)
      • Griffiths-Jones S.
      • Grocock R.J.
      • van Dongen S.
      • Bateman A.
      • Enright A.J.
      miRBase: microRNA sequences, targets and gene nomenclature.
      allowing one mismatch. Novel miRNAs were identified using miRDeep. Total read counts were normalized to read counts per million. Read counts for miRNAs with the same mature sequence were merged. For statistical analysis, we included all unique miRNAs with at least 50 read counts in the sum of all seven samples that is the four samples of HL cell lines and the three samples of GC-B cells. The list with both known and novel miRNAs was further analyzed by GeneSpring GX software version 12.5 (Agilent Technologies, Santa Clara, CA). Significantly differentially expressed miRNAs were identified using a moderated t-test with Benjamini-Hochberg multiple testing correction and a fold change >4. The small RNA sequencing data were deposited in the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo; accession number GSE92616).

      Quantitative Real-Time PCR

      miRNA expression levels were measured using the TaqMan miRNA quantitative PCR assay (Thermo Fisher Scientific Inc.) in a multiplexed manner, as described previously.
      • Kluiver J.
      • Slezak-Prochazka I.
      • van den Berg A.
      Studying microRNAs in lymphoma.
      Based on the small RNA sequencing data, we selected or custom-designed specific TaqMan assays based on the sequence of the most abundant mature miRNA isoforms (Table 1). The miRNA expression levels were normalized to RNU44 (housekeeping gene). Cycle crossing point (Cp) values were determined with the Light Cycler 480 software version 1.5.0 (Roche, Basel, Switzerland). Relative expression levels of miRNAs were determined by calculating 2−ΔCp (ΔCp = CpmiRNA − CpRNU44).
      Table 1The Taqman Assay Catalog Number of miRNAs for Quantitative RT-PCR Validation
      No.miRNATaqMan miRNA assay catalog numberSequence
      1miR-7-18763241023_mat5′-AUCCCGGACGAGCCCCCA-3′
      2miR-9-5p0005835′-UCUUUGGUUAUCUAGCUGUAUGA-3′
      3miR-23a-3p0003995′-AUCACAUUGCCAGGGAUUUCC-3′
      4miR-24-3p0004025′-UGGCUCAGUUCAGCAGGAACAG-3′
      5miR-27a-3p008196_mat5′-UUCACAGUGGCUAAGUUCCG-3′
      6miR-28-5p0004115′-AAGGAGCUCACAGUCUAUUGAG-3′
      7miR-28-3p0024465′-CACUAGAUUGUGAGCUCCUGGA-3′
      8miR-30a-5p006628_mat5′-UGUAAACAUCCUCGACUGGAAGCU-3′
      9miR-92b-3p242469_mat5′-UAUUGCACUCGUCCCGGCCU-3′
      10miR-148a-3p0004705′-UCAGUGCACUACAGAACUUUGU-3′
      11miR-150-5p0004735′-UCUCCCAACCCUUGUACCAGUG-3′
      12miR-190a-5p471572_mat5′-UGAUAUGUUUGAUAUAUUAGGUUG-3′
      13miR-196a-5p241070_mat5′-UAGGUAGUUUCAUGUUGUUGGG-3′
      14miR-301b-3p0023925′-CAGUGCAAUGAUAUUGUCAAAGC-3′
      15miR-320a-3p0022775′-AAAAGCUGGGUUGAGAGGGCGA-3′
      16miR-330-5pCustom assay5′-UCUCUGGGCCUGUGUCUUAGGCU-3′
      17miR-345-5pCustom assay5′-GCUGACUCCUAGUCCAGGGCU-3′
      18miR-363-3p0013555′-AAUUGCACGGUAUCCAUCUGU-3′
      19miR-378a-3p0013145′-ACUGGACUUGGAGUCAGAAGGC-3′
      20miR-615-3p0015885′-UCCGAGCCUGGGUCUCCCUCU-3′
      21let-7b-5p0003785′-UGAGGUAGUAGGUUGUGUGGUU-3′
      22let-7f-2-3pCustom assay5′-CUAUACAGUCUACUGUCUUUCU-3′
      23RNU44 (housekeeping gene)0010945′-CCTGGATGATGATAGCAAATGCTGACTGAACATGAAGGTCTTAATTAGCTCTAACTGACT-3′

      Ago2-RIP–Chromatin IP

      Immunoprecipitation (IP) of the Ago2-containing RISC was performed in four HL cell lines (L1236, L428, L540, and KM-H2), as described previously using 30 million cells as input.
      • Tan L.P.
      • Seinen E.
      • Duns G.
      • de Jong D.
      • Sibon O.C.
      • Poppema S.
      • Kroesen B.J.
      • Kok K.
      • van den Berg A.
      A high throughput experimental approach to identify miRNA targets in human cells.
      Microarray analysis was performed as previously described.
      • Slezak-Prochazka I.
      • Kluiver J.
      • de Jong D.
      • Smigielska-Czepiel K.
      • Kortman G.
      • Winkle M.
      • Rutgers B.
      • Koerts J.
      • Visser L.
      • Diepstra A.
      • Kroesen B.J.
      • van den Berg A.
      Inhibition of the miR-155 target NIAM phenocopies the growth promoting effect of miR-155 in B-cell lymphoma.
      Briefly, cRNA was synthesized from total (T) and IP fractions of four HL cell lines. This was followed by a cRNA amplification and labeling step with cyanine 3-CTP (Cy3) or cyanine 5-CTP (Cy5). Equal amounts of Cy3- or Cy5-labeled cRNA were mixed and hybridized on Human Whole Genome Oligo Microarray overnight (SurePrint G3 Custom GE 8 × 60K; Agilent Technologies). Quantile normalization of signals was performed using GeneSpring GX software version 12.5 (Agilent). Probes with inconsistent intensities in Cy3 and Cy5 replicates and probes not detected in either IP or total fractions were filtered out. For the consistent probes expressed above the background, the average signals for Cy3 and Cy5 replicates were used to calculate IP/T ratio for each sample. The Ago2-RIP–chromatin IP data were deposited in the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo; accession number GSE92615).

      Gene Set Enrichment Analysis

      Gene set enrichment analysis (GSEA version 2.2.0; http://software.broadinstitute.org/gsea/index.jsp) was performed to determine significantly enriched gene sets in Ago2-IP fractions in comparison to total cell lysate fractions.
      • Subramanian A.
      • Tamayo P.
      • Mootha V.K.
      • Mukherjee S.
      • Ebert B.L.
      • Gillette M.A.
      • Paulovich A.
      • Pomeroy S.L.
      • Golub T.R.
      • Lander E.S.
      • Mesirov J.P.
      Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles.
      A total of 8430 gene sets were tested based on Molecular Signatures Database version 5.0 (http://software.broadinstitute.org/gsea/msigdb, last accessed January 10, 2017) using a false discovery rate <0.05. We focused on gene sets enriched in the IP fraction in at least three of four HL cell lines.

      Prediction of Target Genes of miRNAs

      Targetscan version 7.0 (http://www.targetscan.org, last accessed January 10, 2017) was used to generate a list of putative target genes of highly abundant and differentially expressed miRNAs using cumulative weighted context++ scores of genes less than or equal to −0.3.
      • Agarwal V.
      • Bell G.W.
      • Nam J.W.
      • Bartel D.P.
      Predicting effective microRNA target sites in mammalian mRNAs.
      χ2 test was applied to assess whether the percentage of predicted targets of a miRNA was significantly enriched in the Ago2-IP fraction as compared to the percentage in the list of genes expressed in HL cell lines.

      Functional Annotation Analysis

      DAVID bioinformatics Resources version 6.7 (https://david.ncifcrf.gov, last accessed January 10, 2017)
      • Huang da W.
      • Sherman B.T.
      • Lempicki R.A.
      Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.
      was used to functionally annotate genes based on Gene Ontology category of biological process of GOTERM_BP_FAT.

      GFP Competition Assay

      Lentiviral miRZIP constructs to inhibit miR-23a/b-3p, miR-24-3p, and miR-27a/b-3p and a nontargeting control (SCR) were purchased from System Biosciences (Palo Alto, CA). Lentiviral particles were produced in HEK-293T cells by calcium phosphate precipitation transfection. HL cells were infected with miRZIP lentivirus aiming at an infection percentage of 10% to 30%. The cells were cultured for 22 days after infection. The percentage of green fluorescent protein (GFP)+ cells was monitored triweekly by fluorescence-activated cell sorting (BD Biosciences, San Jose, CA). The percentage of GFP-positive cells at day 4 was set to 100%. All GFP competition assays were performed three times.

      Apoptosis Assay

      The percentages of apoptotic cells were determined in L1236 and L428 cells harvested at day 5 and day 8 after transfection with the lentiviral miR-24-3p inhibitor (miRZIP-24-3p) and negative control miRZIP-SCR cells aiming at an infection percentage of >95%. Cells were washed twice with cold phosphate-buffered saline and resuspended at a concentration of 1 × 106 cells/mL in 100 μL calcium buffer (2.6 μg/mL HEPES, 8.18 μg/mL NaCl, and 0.28 μg/mL CaCl2). After staining with Annexin V-APC (BD Biosciences), cells were analyzed by fluorescence-activated cell sorting (BD Biosciences).

      Western Blot

      Cells were lysed in cell lysis buffer (9803; Cell Signaling Technology, Danvers, MA) supplemented with phenylmethylsulfonyl fluoride protease inhibitor. Lysates were kept on ice for 30 minutes and centrifuged at 20,817 × g for 15 minutes at 4°C and supernatant was collected. Protein concentration was measured using the BCA Protein Assay Kit (Thermo Fisher Scientific Inc.) following the manufacturer's instructions. Protein (20 μg) was separated on a polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was incubated overnight at 4°C with primary antibodies diluted in 5% milk in Tris-buffered saline with Tween-20 with anti-BCL2L11 (1000× dilution; C34C5; rabbit monoclonal antibody; Cell Signaling Technology), anti–cyclin-dependent kinase inhibitor 1B [CDKN1B; 100× dilution; P27 (C-19) sc-528; rabbit polyclonal antibody; Santa Cruz Biotechnology, Dallas, TX], anti-MYC (5000× diluted; 1472-1; rabbit monoclonal antibody; Epitomics, Burlingame, CA), anti–sphingosine-1-phosphate receptor 1 (S1PR1; 1000× diluted; ab125074; rabbit monoclonal antibody; Abcam, Cambridge, UK), anti-CARD10 (500× diluted; SAB2702056; rabbit polyclonal antibody; Sigma, Dorset, UK), and anti-Ago2 (1000× diluted; 2E12-1C9; Abnova, Taipei City, Taiwan). A secondary incubation step was performed with goat anti-rabbit or rabbit anti-mouse antibodies (1000× diluted; Dako, Glostrup Municipality, Denmark) conjugated with horseradish peroxidase. Chemoluminescene was detected with Chemi Doc MP scanner and proteins were visualized and quantified with Image Lab software version 4.0.1 (BioRad, Hercules, CA).

      Immunohistochemistry

      Generation of the HL tissue microarrays has been described previously.
      • Xu C.
      • Plattel W.
      • van den Berg A.
      • Ruther N.
      • Huang X.
      • Wang M.
      • de Jong D.
      • Vos H.
      • van Imhoff G.
      • Viardot A.
      • Moller P.
      • Poppema S.
      • Diepstra A.
      • Visser L.
      Expression of the c-Met oncogene by tumor cells predicts a favorable outcome in classical Hodgkin's lymphoma.
      A total of 51 primary diagnostic paraffin-embedded cHL tissue samples were stained with thymus and activation regulated chemokine (TARC; 50× diluted; AF364; polyclonal goat IgG antibody; R&D Systems, Abingdon, UK), anti-CDKN1B [100× dilution; P27 (C-19) sc-528; rabbit polyclonal antibody; Santa Cruz Biotechnology], and anti-MYC (50× diluted; 1472-1; rabbit monoclonal antibody; Epitomics). Antigen retrieval was performed in 0.1 mol/L citrate (pH 6.0) for TARC and Tris EDTA buffer (pH 8.0) in a pressure cooker for 15 minutes. Tissue sections were incubated with primary antibodies for 1 hour at room temperature. The binding was visualized after with 3,3′-diaminobenzidine after second and third antibody incubation steps. Staining with TARC on subsequent tissue sections was used to identify HRS tumor cells. Only cores with ≥10 tumor cells were included in the analysis of CDKN1B/P27kip1 and MYC protein expression patterns in HL.

      Statistical Analysis

      Differential expression of miRNAs between HL cell lines and GC-B cells by quantitative RT-PCR (RT-qPCR) was established using the nonparametric U-test (GraphPad Software Inc., San Diego, CA). For the GFP competition assay, the decrease in percentages of GFP-positive cells in the HL cell lines infected by a miRNA inhibitor (miR-23a/b-3p, miR-24-3p, or miR-27a/b-3p) over time was compared with that of miRZIP-SCR using a mixed model with time and the interaction of time and miRNA inhibitor type as fixed effect and measurement repeat within miRNA inhibitor type as random effect in SPSS (22.0.0.0 version; IBM, Armonk, New York, NY). In case of a nonlinear relation between time and decrease of GFP-positive cell percentages (determined by visual inspection of the graph), quadratic terms for time itself and for time interacting with miRNA inhibitor type were added to the model. No (fixed and random) intercept was included in the model, as the percentages at time 0 were set to 100% and percentages −100% were analyzed. The interaction term is the parameter of interest, as this identifies whether the decrease over time differs between the miRNA and SCR. P < 0.05 was considered statistically significant.

      Results

      miRNA Profiling by Small RNA Sequencing

      An overview of the total number of reads and percentages of mapped reads is given in Supplemental Table S1. The miRNA expression patterns were determined in four cHL cell lines (L1236, L428, L540, and KM-H2), and GC-B cells sorted from three independent tonsils. The top 10 most abundantly expressed miRNAs in HL cells and GC-B cells showed an overlap of six miRNAs (Figure 1). In HL, the top 10 miRNAs were known, whereas in GC-B cells nine known and one novel miRNA were observed. The expression level of the top 10 most abundant miRNAs accounted for 61% of all reads in HL and for 70% in GC-B cells.
      Figure thumbnail gr1
      Figure 1The top 10 most abundant miRNAs in Hodgkin lymphoma (HL) and germinal center (GC)-B cells. A: The top 10 most abundant miRNAs in HL cell lines; these most abundant miRNAs account for 61% of all reads. B: The top 10 most abundant miRNAs in GC-B cells, accounting for 70% of all reads. Asterisks indicate miRNAs present in the top 10 of both HL cell lines and GC-B cells.
      A total of 84 miRNAs were significantly differentially expressed between HL cells and GC-B cells, including 55 up-regulated (54 known and 1 novel) and 29 down-regulated (26 known and 3 novel) miRNAs (Figure 2A). None of the 10 most abundant miRNAs in HL were significantly differentially expressed between HL and GC-B cells.
      Figure thumbnail gr2
      Figure 2Deregulated miRNAs in Hodgkin lymphoma (HL) as compared to germinal center (GC)-B cells. A: Heat map of 84 differentially expressed miRNAs identified by a moderated t-test and a fold change >4 between HL cell lines and GC-B cells. B: Quantitative RT-PCR validation of 12 miRNAs with increased expression levels shows significant differences in the expected direction for 11 miRNAs. C: Quantitative RT-PCR validation of seven miRNAs with decreased expression levels reveals a significant difference for four miRNAs. P < 0.05 (U-test).
      A total of 15 up-regulated and seven down-regulated miRNAs were selected for validation by RT-qPCR. The expression levels of 3 of 15 up-regulated miRNAs were lower than the detection limit. For 11 of 15 miRNAs significantly increased levels were observed in HL cell lines compared to GC-B cells (ie, miR-9-5p, miR-23a-3p, miR-24-3p, miR-27a-3p, miR-92b-3p, miR-196a-5p, miR-301b-3p, miR-320a-3p, miR-345-5p, miR-378a-3p, and miR-615-3p) (Figure 2B). Unexpectedly, a significant decrease in let-7b-5p levels was observed in HL, a pattern opposite to that observed with small RNA sequencing (Figure 2B). For four of seven down-regulated miRNAs a significantly decreased expression level was confirmed in HL cell lines compared to GC-B cells (ie, miR-28-5p, miR-148a-3p, miR-150-5p, and miR-363-5p) (Figure 2C). For miR-28-3p, miR-30a-5p, and miR-7-18763, the down-regulation could not be validated. These results showed that the differential expression pattern could be confirmed for most of the miRNAs.

      miR-24-3p and miR-27-3p Act as Oncogenes in HL

      miR-23, miR-24, and miR-27 are transcribed from two primary miRNA transcripts (ie, C9orf3, located at chromosome 9, and LOC284453, located at chromosome 19). C9orf3 is the primary (pri-)miRNA transcript of miR-23b, miR-27b, and miR-24-1 and LOC284454 is the pri-miRNA transcript of miR-24-2, miR-27a, and miR-23a. miR-27b-3p was in the top 10 most abundantly expressed miRNAs; miR-23a-3p, miR-24-3p, and miR-27a-3p were differentially expressed. To study the functional relevance of these miRNAs on HL cell growth, we inhibited miR-23a/b-3p, miR-24-3p, and miR-27a/b-3p using specific miRNA inhibitors (miRZIP) in four HL cell lines. We observed a significant decrease over time in the percentage of GFP-positive cells for miRZIP-24-3p compared to the control in L1236, L428, and KM-H2 (Figure 3A). A significant decrease in GFP-positive cells was seen for miRZIP-27a-3p in L1236 and L540 as well as for miRZIP-27b-3p in L1236 (Supplemental Figure S1). For miRZIP-23a-3p we found either no effect or a significant increase (in L540) possibly because of a continued decrease of the percentage of GFP+ cells in miRZIP-SCR infected cells (Supplemental Figure S1).
      Figure thumbnail gr3
      Figure 3The effect of miR-24-3p inhibition on cell growth and apoptosis. A: Green fluorescent protein (GFP) competition assay of miR-24-3p inhibitor (miRZIP-24-3p) and control miRZIP-SCR–infected Hodgkin lymphoma (HL) cell lines L1236, L428, L540, and KM-H2. MiRZIP-24-3p was stably transfected in cells using a viral vector, which coexpresses GFP. The GFP percentage was measured triweekly for 22 days, and the percentage at the first day of measurement (day 4) was set to 1. Significant differences were calculated using a mixed model analysis. B: The percentage of apoptotic cells on inhibition of miR-24-3p in L1236 and L428 was assessed by determining the percentage of annexin V–positive cells at day 5 and day 8 after lentiviral infection in duplicate. Data are expressed as means ± SD (A). n = 3 (A). P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus miRZIP-SCR (paired t-test).
      To study the cause of the decrease in the percentages of GFP-positive cells, we analyzed cell cycle distribution and the presence of apoptotic cells in L1236 and L428 at day 5 and day 8 on miR-24-3p inhibition. We found no effect on the distribution of the cell cycle phases (data not shown). The percentage of apoptotic cells based on annexin V staining was significantly increased in both L1236 and L428 on inhibition of miR-24-3p (Figure 3B). In line with a stronger decrease in cell growth in L1236, the increase of apoptotic cells was also the highest in L1236. Thus, inhibition of miR-24-3p leads to an increase of apoptotic cells in HL, providing an explanation for the decrease in GFP+ cells in the growth competition assay.

      Identification of miRNA Target Genes by Ago2-RIP–Chromatin IP

      IP with anti-Ago2 was performed to pull down mRNA transcripts of miRNA target genes in HL cell lines. The efficiency of the IP procedure was validated by Western blot and RT-qPCR. Ago2 protein was detected in the Ago2-IP and the total cell lysate fractions but not in flow through (Supplemental Figure S2A). In the control IgG-IP samples, Ago2 protein was present in the total and flow through fractions but not in the IP. Using RT-qPCR, we observed a strong enrichment of two randomly selected highly expressed miRNAs in the Ago2-IP but not in the IgG-IP fractions (Supplemental Figure S2B). Using gene set enrichment analysis, we identified significant enrichment of multiple miRNA target gene sets in all four HL cell lines. The miRNA gene sets of top 10 miRNAs in HL were all significantly enriched in the Ago2-IP (Supplemental Table S2). Of the 17 in HL up-regulated miRNAs with the highest read frequencies (at least 200 read counts per million on average), 12 showed significant enrichment of their miRNA target gene sets. For the remaining miRNAs, no target gene sets were available for three, whereas no significant enrichment was observed for two (Supplemental Table S2). All together, these data confirmed the efficiency of the Ago2-IP enrichment procedure.
      To define the set of genes regulated by miRNAs in HL, we determined the Ago2-IP over total signal intensity ratios of all probes. The number of probes enriched in the Ago2-IP fraction (IP/T >2) was 2080 in KM-H2, 3230 in L1236, 2107 in L428, and 2171 in L540 (Figure 4A). Using a threshold of having an IP/T >2 in at least three of four cell lines, we identified 1142 unique protein-coding genes (represented by 1434 probes) that together represent the HL miRNA-targetome (Figure 4A and Supplemental Table S3).
      Figure thumbnail gr4
      Figure 4Identification of target genes of each miRNA family. A: Ago2-IP–enriched mRNA probes per cell line (fold change of IP/total >2) and the 1434 probes (1142 genes) that are IP-enriched in at least three of the four Hodgkin lymphoma (HL) cell lines (red cross). B: Comparison of the percentage of Targetscan-predicted target genes among the expressed and Ago2-IP–enriched of five miRNAs with low expression levels in HL. C: Comparison of the percentage of Targetscan-predicted target genes among the expressed and Ago2-IP enriched of 11 differentially expressed and validated miRNAs. P < 0.05, ∗∗∗P < 0.001 (χ2 test).
      We next determined whether the putative target genes of the 11 validated up-regulated miRNAs were enriched in the HL miRNA targetome. For eight miRNAs, we observed significant enrichment of target genes in the Ago2-IP fractions (Figure 4C and Table 2). For the remaining three, few of the predicted targets were expressed in HL. As negative control, we also analyzed enrichment of five miRNAs with low or no expression in HL (ie, miR-342-5p, miR-4488-5p, miR-3150b-3p, miR-150-3p, and miR-6500-3p). No enrichment was seen for four of these miRNAs (Figure 4B). For miR-150-3p, we observed a moderate but significant increase of predicted target gene set, although the expression of this miRNA is low in HL (average less than seven reads per million). This is probably caused by the high overlap of putative miR-150-3p target genes with targets of one or more of the top 10 most abundant miRNAs in HL (12/25 genes).
      Table 2Ago2-Immunoprecipitation Data Analysis of miRNAs Up-Regulated in Hodgkin Lymphoma
      miRNAsQuantitative RT-PCR validationAgo2-IP
      miRNA gene sets enrichedmiRNA targets enriched
      hsa-miR-9-5pYesYesYes
      hsa-miR-23a-3pYesYesYes
      hsa-miR-24-3pYesYesYes
      hsa-miR-27a-3pYesYesYes
      hsa-miR-92b-3pYesYesYes
      hsa-miR-196a-5pYesYesYes
      hsa-miR-301b-3pYesYesYes
      hsa-miR-320aYesNoYes
      hsa-miR-345-5pYesNoNo
      hsa-miR-378a-3pYesNoNo
      hsa-miR-615-3pYesNANo
      hsa-let-7b-5pNoYesYes
      NA, not applicable.

      CDKN1B/P27kip1 and MYC Are Targets of miR-24-3p

      To identify target genes that might be relevant for the observed miR-24-3p phenotype, we first determined how many predicted and/or validated miR-24-3p target genes were Ago2-IP enriched in the HL cell lines. This revealed a total of 52 putative miR-24-3p target genes among the 1142 consistently Ago2-IP enriched genes (Supplemental Table S4). Functional annotation analysis revealed gene ontology related to cell growth and apoptosis for 15 of these targets (Table 3). Six of the 15 putative miR-24-3p targets (ie, CDKN1B,
      • Giglio S.
      • Cirombella R.
      • Amodeo R.
      • Portaro L.
      • Lavra L.
      • Vecchione A.
      MicroRNA miR-24 promotes cell proliferation by targeting the CDKs inhibitors p27Kip1 and p16INK4a.
      • Lynch S.M.
      • McKenna M.M.
      • Walsh C.P.
      • McKenna D.J.
      miR-24 regulates CDKN1B/p27 expression in prostate cancer.
      SIPR1,
      • Lorenzen J.M.
      • Kaucsar T.
      • Schauerte C.
      • Schmitt R.
      • Rong S.
      • Hubner A.
      • Scherf K.
      • Fiedler J.
      • Martino F.
      • Kumarswamy R.
      • Kolling M.
      • Sorensen I.
      • Hinz H.
      • Heineke J.
      • van Rooij E.
      • Haller H.
      • Thum T.
      MicroRNA-24 antagonism prevents renal ischemia reperfusion injury.
      CARD10,
      • Zhang S.
      • Zhang C.
      • Liu W.
      • Zheng W.
      • Zhang Y.
      • Wang S.
      • Huang D.
      • Liu X.
      • Bai Z.
      MicroRNA-24 upregulation inhibits proliferation, metastasis and induces apoptosis in bladder cancer cells by targeting CARMA3.
      BCL2L11,
      • Zhang H.
      • Duan J.
      • Qu Y.
      • Deng T.
      • Liu R.
      • Zhang L.
      • Bai M.
      • Li J.
      • Ning T.
      • Ge S.
      • Wang X.
      • Wang Z.
      • Fan Q.
      • Li H.
      • Ying G.
      • Huang D.
      • Ba Y.
      Onco-miR-24 regulates cell growth and apoptosis by targeting BCL2L11 in gastric cancer.
      MYC,
      • Lal A.
      • Navarro F.
      • Maher C.A.
      • Maliszewski L.E.
      • Yan N.
      • O'Day E.
      • Chowdhury D.
      • Dykxhoorn D.M.
      • Tsai P.
      • Hofmann O.
      • Becker K.G.
      • Gorospe M.
      • Hide W.
      • Lieberman J.
      miR-24 inhibits cell proliferation by targeting E2F2, MYC, and other cell-cycle genes via binding to “seedless” 3′UTR microRNA recognition elements.
      and INSIG1)
      • Ng R.
      • Wu H.
      • Xiao H.
      • Chen X.
      • Willenbring H.
      • Steer C.J.
      • Song G.
      Inhibition of microRNA-24 expression in liver prevents hepatic lipid accumulation and hyperlipidemia.
      were validated previously. Based on average fold changes in the Ago2-IP/T fractions, the top five genes were selected for further analysis by Western blot. CDKN1B/P27kip1 and S1PR1 contained one conserved 8-mer binding site in the 3′-untranslated region, BCL2 like 11 (BCL2L11) contained two conserved 8-mer sites, CARD10 contained two poorly conserved 7-mer-m8 binding sites, and MYC contained one poorly conserved 7-mer-A binding site. The endogenous protein level of CDKN1B/P27kip1 was lower in KM-H2 compared to the three other cell lines (Supplemental Figure S3A). MYC levels were low in L1236 (Supplemental Figure S3B) and higher in the other cell lines. The levels of BCL2L11 were the highest in L1236 and low in KM-H2 and L540 (Supplemental Figure S3C). The S1PR1 protein levels were highest in L428 and slightly lower in the other cell lines (Supplemental Figure S3D). CARD10 protein levels were lower than the detection limit in all four HL cell lines (data not shown).
      Table 3Fifteen Ago2-IP–Enriched Predicted miR-24-3p Target Genes Related to Cell Growth
      No.Putative targetsGene nameFC of IP/total in Ago2-IPConserved BS (Targetscan)References
      KMH2L1236L428L540AvgScore8-mer7-mer
      1CDKN1BCyclin-dependent kinase inhibitor 1B (p27, Kip1)7.6019.437.9510.5211.38−0.5310
      • Giglio S.
      • Cirombella R.
      • Amodeo R.
      • Portaro L.
      • Lavra L.
      • Vecchione A.
      MicroRNA miR-24 promotes cell proliferation by targeting the CDKs inhibitors p27Kip1 and p16INK4a.
      • Lynch S.M.
      • McKenna M.M.
      • Walsh C.P.
      • McKenna D.J.
      miR-24 regulates CDKN1B/p27 expression in prostate cancer.
      2S1PR1Sphingosine-1-phosphate receptor 110.1116.862.498.429.47−0.3910
      • Lorenzen J.M.
      • Kaucsar T.
      • Schauerte C.
      • Schmitt R.
      • Rong S.
      • Hubner A.
      • Scherf K.
      • Fiedler J.
      • Martino F.
      • Kumarswamy R.
      • Kolling M.
      • Sorensen I.
      • Hinz H.
      • Heineke J.
      • van Rooij E.
      • Haller H.
      • Thum T.
      MicroRNA-24 antagonism prevents renal ischemia reperfusion injury.
      3CARD10Caspase recruitment domain family, member 107.416.82NA9.207.81−0.3000
      • Zhang S.
      • Zhang C.
      • Liu W.
      • Zheng W.
      • Zhang Y.
      • Wang S.
      • Huang D.
      • Liu X.
      • Bai Z.
      MicroRNA-24 upregulation inhibits proliferation, metastasis and induces apoptosis in bladder cancer cells by targeting CARMA3.
      4BCL2L11BCL2-like 11 (apoptosis facilitator)4.747.443.48NA5.22−0.7220
      • Zhang H.
      • Duan J.
      • Qu Y.
      • Deng T.
      • Liu R.
      • Zhang L.
      • Bai M.
      • Li J.
      • Ning T.
      • Ge S.
      • Wang X.
      • Wang Z.
      • Fan Q.
      • Li H.
      • Ying G.
      • Huang D.
      • Ba Y.
      Onco-miR-24 regulates cell growth and apoptosis by targeting BCL2L11 in gastric cancer.
      5MYCV-mycmyelocytomatosis viral oncogene homolog (avian)2.964.036.721.765.16NA00
      • Lal A.
      • Navarro F.
      • Maher C.A.
      • Maliszewski L.E.
      • Yan N.
      • O'Day E.
      • Chowdhury D.
      • Dykxhoorn D.M.
      • Tsai P.
      • Hofmann O.
      • Becker K.G.
      • Gorospe M.
      • Hide W.
      • Lieberman J.
      miR-24 inhibits cell proliferation by targeting E2F2, MYC, and other cell-cycle genes via binding to “seedless” 3′UTR microRNA recognition elements.
      6TNFSF9Tumor necrosis factor (ligand) superfamily, member 92.885.334.47NA4.23−0.4101
      7PLK3Polo-like kinase 3 (Drosophila)2.174.454.156.064.21−0.3101
      8APPL2Adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 22.923.58NA5.884.13−0.3910
      9SESN1Sestrin 11.825.832.775.223.91−0.3801
      10CISHCytokine inducible SH2-containing protein4.353.743.093.533.68−0.4900
      11MNTMAX binding protein3.124.673.001.683.12−0.6111
      12INSIG1Insulin induced gene 12.164.462.132.082.71−0.3003
      • Ng R.
      • Wu H.
      • Xiao H.
      • Chen X.
      • Willenbring H.
      • Steer C.J.
      • Song G.
      Inhibition of microRNA-24 expression in liver prevents hepatic lipid accumulation and hyperlipidemia.
      13KIF5AKinesin family member 5ANA2.852.242.632.57−0.3100
      14BBC3BCL2 binding component 32.243.362.491.712.45−0.5312
      15PIM2Pim-2 oncogene1.182.412.152.221.99−0.5102
      Score: Cumulative weighted context ++ score in Targetscan. Putative targets were sorted based on average FC (avg) in bold. References are given for all previously validated target genes of miR-24-3p.
      Avg, average; BS, binding site; FC, fold change; IP, immunoprecipitation; NA, not applicable.
      To establish a possible effect of miR-24-3p on the expression of the four proteins expressed in HL, we analyzed their levels on inhibition of miR-24-3p in L1236 and L428. Inhibition of miR-24-3p had no effects on BCL2L11 and S1PR1 protein levels in either L1236 or L428 (Supplemental Figure S3, E and F). CDKN1B/P27kip1 and MYC protein levels were consistently increased in miRZIP-24-3p–infected L1236 cells compared to control miRZIP-SCR infected cells. In contrast, no changes in protein levels were observed in L428 cells (Figure 5A). To further explore the relevance of CDKN1B/P27kip1 and MYC as targets of miR-24-3p in HL, we analyzed two additional HL cell lines (ie, HDLM2 and SUPHD1). Inhibition of miR-24-3p induced a reduction in cell growth similar to the other HL cell lines (Figure 5C). Moreover, an increase in CDKN1B/P27kip1 protein levels on inhibition of miR-24-3p was observed in both cell lines, whereas an increase in MYC protein levels was observed only in HDLM2 (Figure 5B).
      Figure thumbnail gr5
      Figure 5CDKN1B/P27kip1 and MYC are target genes of miR-24-3p in Hodgkin lymphoma (HL). A: Effect of miR-24-3p inhibition on the protein levels of CDKN1B/P27kip1 and MYC in L1236 and L428. Cells were harvested at day 5 after infection with miRZIP-24-3p and miRZIP-SCR in one of two independent experiments. B: miR-24-3p inhibition effects on the protein levels of CDKN1B/P27kip1 and MYC in HDLM2 and SUPHD1. Cells were harvested at day 5 for HDLM2 and day 10 for SUPHD1 after infection in one experiment. Protein levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). C: GFP competition assay of miRZIP-24-3p and miRZIP-SCR infection on HDLM2 and SUPHD1 HL cell lines. D: Representative HL case showing expression of CDKN1B/P27kip1 and MYC in HRS tumor cells (arrows). Data are expressed as means ± SD (C). n = 3 (C). ∗∗∗P < 0.001 versus miRZIP-SCR (mixed model analysis). Original magnification, ×400 (D).
      To evaluate protein expression in HRS cells, we analyzed their expression in HL tissue samples. CDKN1B/P27kip1 (46/46 = 100%) and MYC (42/46 = 91%) were expressed in most HL cases (Figure 5D and Supplemental Table S3). The percentages of CDKN1B/P27kip1 positive tumor cells varied from 5% to 100% and of MYC varied from 0% to 90%. Staining in >50% of the tumor cells was observed in 26 of 46 for CDKN1B/P27kip1 and in 22 of 46 for MYC. No obvious correlation was observed between CDKN1B/P27kip1 and MYC protein expression in HRS cells of HL cases.

      Discussion

      In this study, we found a total of 84 significant differentially expressed miRNAs in HL cell lines compared to GC-B cells. Three of these miRNAs (ie, miR-23a-3p, miR-24-3p, and miR-27a-3p) were transcribed from a single primary miRNA transcript. miR-24-3p inhibition impaired the cell growth in five of six cell lines, by increasing the number of apoptotic cells. Moreover, 52 of 1142 Ago2-IP enriched genes were predicted miR-24-3p targets. CDKN1B/P27kip1 and MYC protein levels were regulated by miR-24-3p in HL cell lines and both proteins were expressed in a variable percentage of the tumor cells in primary HL tissue samples.
      Validation of the small RNA sequencing data by RT-qPCR revealed consistent results for most of them. Some of the miRNAs showed a different pattern by RT-qPCR. For miR-30a-5p, this might be caused by high expression of other miRNAs of the same seed family (miR-30a/b/c/d/e-5p). The mature miRNA sequences of miR-30d-5p and miR-30e-5p differ by only one nucleotide from miR-30a-5p. The expression of miR-30d-5p and miR-30e-5p is more abundant in HL and these miRNAs have similar expression levels in GC-B cells (data not shown). So, this might explain the difference between small RNA sequencing and RT-qPCR results. For let-7-5p, miR-28-3p, and the novel miRNA miR-7-18763, we could not explain the differences between the two techniques. For let-7-5p, this is unexpected as all eight let-7-5p family members show the same pattern with higher read counts in HL compared to GC-B cells.
      Based on the reduced growth of HL cells on inhibition of miR-24-3p, we concluded that this miRNA might have an oncogenic role in HL. Several other studies also revealed an oncogenic role for miR-24-3p in other types of cancers, including gastric cancer,
      • Zhang H.
      • Duan J.
      • Qu Y.
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      • Liu R.
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      • Wang Z.
      • Fan Q.
      • Li H.
      • Ying G.
      • Huang D.
      • Ba Y.
      Onco-miR-24 regulates cell growth and apoptosis by targeting BCL2L11 in gastric cancer.
      pancreatic carcinoma,
      • Liu R.
      • Zhang H.
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      • Ba Y.
      The miR-24-Bim pathway promotes tumor growth and angiogenesis in pancreatic carcinoma.
      and tongue squamous cell carcinoma.
      • Zheng X.
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      MicroRNA-24 induces cisplatin resistance by targeting PTEN in human tongue squamous cell carcinoma.
      On the other hand, tumor suppressor activity has also been reported for miR-24-3p in breast cancer, cervical cancer,
      • Srivastava N.
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      • Bamezai R.N.
      miR-24-2 controls H2AFX expression regardless of gene copy number alteration and induces apoptosis by targeting antiapoptotic gene BCL-2: a potential for therapeutic intervention.
      and gastric cancer.
      • Qin W.
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      miR-24 regulates apoptosis by targeting the open reading frame (ORF) region of FAF1 in cancer cells.
      Nie et al
      • Nie K.
      • Gomez M.
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      • Garcia J.F.
      • Liu Y.
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      • Tam W.
      MicroRNA-mediated down-regulation of PRDM1/Blimp-1 in Hodgkin/Reed-Sternberg cells: a potential pathogenetic lesion in Hodgkin lymphomas.
      showed abundant expression of miR-24-3p in HL cell lines; however, in our study, the levels were more average (average rank, 84 of 453 miRNAs expressed). This difference might be caused by differences in experimental procedures. The previous study used cloning of miRNAs, followed by sequencing, and mapped their miRNAs to miRBASE version 9.1 (version 9.1 contains 4449 entries).
      • Nie K.
      • Gomez M.
      • Landgraf P.
      • Garcia J.F.
      • Liu Y.
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      • Tam W.
      MicroRNA-mediated down-regulation of PRDM1/Blimp-1 in Hodgkin/Reed-Sternberg cells: a potential pathogenetic lesion in Hodgkin lymphomas.
      We used small RNA sequencing and mapped our miRNAs to miRBASE version 21 (version 21 contains 28,645 entries).
      Of the top five putative target genes of miR-24-3p based on Ago2-IP analysis, we found no protein expression for CARD10, whereas levels of BCL2L11 and S1PR1 did not change on miR-24-3p inhibition. For the other two top five targets, we observed an increase in protein level on inhibition of miR-24-3p. For CDKN1B/P27kip1, protein levels increased in three of four HL cell lines, and for MYC, protein levels increased in two of four cell lines. CDKN1B/P27kip1 belongs to the Cip/Kip family of CDK inhibitor proteins. CDKN1B/P27kip1 prevents activation of the cyclin E–CDK2 complex. By phosphorylating Rb, this complex regulates the release of E2Fs, transcription factors with important functions in the control of cell cycle progression and apoptosis.
      • Polyak K.
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      p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-beta and contact inhibition to cell cycle arrest.
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      E2F1 as a target: promoter-driven suicide and small molecule modulators.
      This implicates that high miR-24-3p levels possibly promote cell growth and protect from apoptosis by inhibiting CDKN1B/P27kip1 in HL (Figure 6). MYC is a transcription factor that can both activate and repress its target genes. MYC target genes have been shown to play a role in cell cycle, apoptosis, and cellular transformation.
      • Dang C.V.
      c-Myc target genes involved in cell growth, apoptosis, and metabolism.
      On the one hand, overexpression of MYC has been associated with development of many types of cancers.
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      The myc oncogene: its role in transformation and differentiation.
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      Association with c-Myc: an alternated mechanism for c-Myc function.
      In line with this, it was shown that knockdown of MYC increased apoptosis in L1236, L428, and L540 HL cell lines.
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      The two HL cell lines that showed an increase in MYC on miR-24-3p inhibition (L1236 and HDLM2) and one of the cell lines that did not show an increase in MYC on miR-24-3p inhibition (L428) had a mutation in the p53 gene.
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      Thus, in HL, the increase in apoptosis on miR-24-3p inhibition seems not to be related to the p53 mutational status. Others have shown that induction or sensitization that leads to apoptosis on high levels of MYC can also be p53 independent.
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      MYC can sensitize cells to CD95/Fas-mediated apoptosis
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      and amplify the mitochondrial apoptotic pathway by inhibiting antiapoptotic members of the BCL2 family and activating proapoptotic BCL2 members.
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      c-Myc overexpression sensitizes Bim-mediated Bax activation for apoptosis induced by histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) through regulating Bcl-2/Bcl-xL expression.
      Thus, we speculate that both increased and decreased MYC levels might negatively affect HL cell growth.
      Figure thumbnail gr6
      Figure 6Model of the oncogenic action of miR-24-3p in Hodgkin lymphoma (HL). High miR-24-3p levels result in reduced CDKN1B/P27kip1 levels. Loss of this negative regulator leads to activation of CDK2/Cyclin E, which leads to phosphorylation of the retinoblastoma protein (Rb) and release of the E2F transcription factor. Downstream E2F promote proliferation and inhibit apoptosis. High miR-24-3p levels also lead to reduced MYC protein levels, which might contribute to decreased sensitivity to apoptosis. Thus, miR-24-3p plays a role in growth and survival of HL cells by regulating the protein levels of CDKN1B/P27kip1 and MYC.
      Immunohistochemistal staining for CDKN1B/P27kip1 and MYC in primary HL tissues did not reveal a clear correlation between their expression levels. This implicated that miR-24-3p was not the only factor regulating their expression levels. It might be that other miRNAs target one, but not the other gene, which might explain the lack of correlation between these two proteins. If such a miRNA is variably expressed in HL, then CDKN1B/P27kip1 and MYC repression will not only vary based on miR-24 expression. The expression of CDKN1B/P27kip1 and MYC might also differ because of changes in the presence of other regulators, such as specific transcription factors or different epigenetic marks.
      In summary, we showed a specific miRNA expression profile in HL and characterized the HL miRNA targetome. Inhibition of the oncogenic miR-24-3p induced apoptosis, possibly via targeting CDKN1B/P27kip1 and MYC.

      Acknowledgments

      We thank Geert Mesander and Henk Moes for flow cytometry assistance and members from the Department of Otorhinolaryngology/Head and Neck Surgery (University Medical Center Groningen, Groningen, the Netherlands) for their help with the collection of tonsil tissues.
      Y.Y., J.Ko., D.d.J., B.R., and M.T. performed research; Y.Y., J.Kl., I.M.N., A.D., L.V., and K.K. analyzed data; B.P. contributed germinal center B-cell samples; Y.Y., J.Kl., and A.v.d.B. designed the study and wrote the manuscript.

      Supplemental Data

      • Supplemental Figure S1

        Green fluorescent protein (GFP) competition assays on miR-23a/b-3p and miR-27a/b-3p inhibition in Hodgkin lymphoma (HL) cell lines. GFP competition assay using miR-23a-3p, miR-27b-3p (miRZIP-23a-3p and miRZIP-27b-3p) (A) and miR-23b-3p and miR-27a-3p inhibitors (miRZIP-23a-3p and miRZIP-27b-3p) (B) in L1236, L428, L540, and KM-H2 HL cell lines. miRNA inhibitor (miRZIP) constructs that coexpressed GFP were transduced to HL cells. The GFP percentage was measured triweekly for 22 days. P < 0.05, ∗∗∗P < 0.001 versus miRZIP-SCR.

      Figure thumbnail figs1
      Supplemental Figure S2Control of the Ago2-IP efficiency in four Hodgkin lymphoma (HL) cell lines. A: The immunoprecipitated fraction (IP) efficiency was validated by Western blot. An antibody against IgG was used as a negative control. Ago2 protein was effectively pulled down in Ago2-IP fraction in all four HL cell lines. Arrows indicate the Ago2 protein. B: The IP efficiency was validated by quantitative RT-PCR. The levels of miR-17-5p and miR-92a-3p were strongly enhanced in the Ago2-IP fraction as compared to the control IgG-IP fraction. FT, flow-through fraction; T, total cell lysate.
      Figure thumbnail figs2
      Supplemental Figure S3Effects of miR-24-3p inhibition on BCL2L11 and S1PR1 protein levels in L1236 and L428 Hodgkin lymphoma (HL) cell lines. CDKN1B/P27kip1 (A), MYC (B), BCL2L11 (C), and S1PR1 (D) protein levels were measured in HL cell lines L1236, L428, L540, and KM-H2. Effect of miR-24-3p inhibition on the protein levels of BCL2L11 (E) and S1PR1 (F) in L1236 and L428. Cells were harvested at day 5 after infection with miRZIP-24-3p and miRZIP-SCR. Shown is a representative result of one of two independent experiments. Protein levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The protein levels could not be quantified for the smallest isoform of the BCL2L11 protein. EL, extra long; L, long; S, small.

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        c-Myc overexpression sensitizes Bim-mediated Bax activation for apoptosis induced by histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) through regulating Bcl-2/Bcl-xL expression.
        Int J Biochem Cell Biol. 2007; 39: 1016-1025

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        The American Journal of PathologyVol. 189Issue 2
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          In the article entitled, “miR-24-3p Is Overexpressed in Hodgkin Lymphoma and Protects Hodgkin and Reed-Sternberg Cells from Apoptosis” (Volume 187, pages 1343–1355 of the June 2017 issue of The American Journal of Pathology; https://doi.org/10.1016/j.ajpath.2017.02.016), the authors have discovered that an incorrect HDLM2 cell line was possibly used in the original experiments presented in Figure 5, B–C. As the materials used in the original experiments were no longer available to re-test, the authors have replaced the cell line and re-run the experiments, confirming that the correct HDLM2 line was used.
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