MicroRNA-Mediated Down-Regulation of PRDM1/Blimp-1 in Hodgkin/Reed-Sternberg Cells: A Potential Pathogenetic Lesion in Hodgkin Lymphomas
2008; Elsevier BV; Volume: 173; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2008.080009
ISSN1525-2191
AutoresKui Nie, Mario Gómez, Pablo Landgraf, J. F. García, Yifang Liu, Leonard Tan, Amy Chadburn, Thomas Tuschl, Daniel M. Knowles, Wayne Tam,
Tópico(s)RNA modifications and cancer
ResumoPRDM1/Blimp-1, a master regulator in terminal B-cell differentiation, has been recently identified as a tumor suppressor target for mutational inactivation in diffuse large B-cell lymphomas of the activated B-cell type. Our studies here demonstrate that PRDM1/blimp-1 is also a target for microRNA (miRNA)-mediated down-regulation by miR-9 and let-7a in Hodgkin/Reed-Sternberg (HRS) cells of Hodgkin lymphoma (HL). MiRNA expression profiling by direct miRNA cloning demonstrated that both of these miRNAs are among the most highly expressed in cultured HRS cells. These miRNAs functionally targeted specific binding sites in the 3′ untranslated region of PRDM1/blimp-1 mRNA and repressed luciferase reporter activities through repression of translation. In addition, high levels of miR-9 and let-7a in HL cell lines correlated with low levels of PRDM1/Blimp-1. Similar to their in vitro counterparts, the majority of HRS cells in primary HL cases showed weak or no PRDM1/Blimp-1 expression. Over-expression of miR-9 or let-7a reduced PRDM1/Blimp-1 levels in U266 cells by 30% to 50%, whereas simultaneous inhibition of their activities in L428 cells resulted in an approximately 2.6-fold induction in PRDM1/Blimp-1. MiRNA-mediated down-regulation of PRDM1/Blimp-1 may contribute to the phenotype maintenance and pathogenesis of HRS cells by interfering with normal B-cell terminal differentiation, thus representing a novel molecular lesion, as well as a potential therapeutic target in HL. PRDM1/Blimp-1, a master regulator in terminal B-cell differentiation, has been recently identified as a tumor suppressor target for mutational inactivation in diffuse large B-cell lymphomas of the activated B-cell type. Our studies here demonstrate that PRDM1/blimp-1 is also a target for microRNA (miRNA)-mediated down-regulation by miR-9 and let-7a in Hodgkin/Reed-Sternberg (HRS) cells of Hodgkin lymphoma (HL). MiRNA expression profiling by direct miRNA cloning demonstrated that both of these miRNAs are among the most highly expressed in cultured HRS cells. These miRNAs functionally targeted specific binding sites in the 3′ untranslated region of PRDM1/blimp-1 mRNA and repressed luciferase reporter activities through repression of translation. In addition, high levels of miR-9 and let-7a in HL cell lines correlated with low levels of PRDM1/Blimp-1. Similar to their in vitro counterparts, the majority of HRS cells in primary HL cases showed weak or no PRDM1/Blimp-1 expression. Over-expression of miR-9 or let-7a reduced PRDM1/Blimp-1 levels in U266 cells by 30% to 50%, whereas simultaneous inhibition of their activities in L428 cells resulted in an approximately 2.6-fold induction in PRDM1/Blimp-1. MiRNA-mediated down-regulation of PRDM1/Blimp-1 may contribute to the phenotype maintenance and pathogenesis of HRS cells by interfering with normal B-cell terminal differentiation, thus representing a novel molecular lesion, as well as a potential therapeutic target in HL. PRDM1, also known as Blimp-1, belongs to the PRDM gene family of transcription repressors containing Kruppel-type zinc fingers and the SET-related PR (PRDI-BF1-RIZ) domain. PRDM1 is expressed as two isoforms, α and β, as a result of alternative promoter usage. They differ in that the latter lacks the amino-terminal acidic domain and part of the PR domain, and is functionally impaired.1Gyory I Fejer G Ghosh N Seto E Wright KL Identification of a functionally impaired positive regulatory domain I binding factor 1 transcription repressor in myeloma cell lines.J Immunol. 2003; 170: 3125-3133PubMed Google Scholar PRDM1 plays a critical role in terminal differentiation of lymphocytes2Kallies A Nutt SL Terminal differentiation of lymphocytes depends on Blimp-1.Curr Opin Immunol. 2007; 19: 156-162Crossref PubMed Scopus (107) Google Scholar and epidermal cells,3Magnusdottir E Kalachikov S Mizukoshi K Savitsky D Ishida-Yamamoto A Panteleyev AA Calame K Epidermal terminal differentiation depends on B lymphocyte-induced maturation protein-1.Proc Natl Acad Sci USA. 2007; 104: 14988-14993Crossref PubMed Scopus (128) Google Scholar as well as cell fate specification of primordial germ cells4Hayashi K de Sousa Lopes SM Surani MA Germ cell specification in mice.Science. 2007; 316: 394-396Crossref PubMed Scopus (243) Google Scholar and other cell types.5Robertson EJ Charatsi I Joyner CJ Koonce CH Morgan M Islam A Paterson C Lejsek E Arnold SJ Kallies A Nutt SL Bikoff EK Blimp1 regulates development of the posterior forelimb, caudal pharyngeal arches, heart and sensory vibrissae in mice.Development. 2007; 134: 4335-4345Crossref PubMed Scopus (103) Google Scholar In B cells, PRDM1 is a key differentiation factor in post-germinal center (GC) cells and is regarded as a master regulator for plasma cell differentiation.6Johnson K Shapiro-Shelef M Tunyaplin C Calame K Regulatory events in early and late B-cell differentiation.Mol Immunol. 2005; 42: 749-761Crossref PubMed Scopus (53) Google Scholar In normal lymphoid tissues, PRDM1 is co-expressed with interferon regulatory factor-4 (IRF4) in plasma cells and in a subset of GC cells that demonstrate evidence of plasma cell commitment and differentiation.7Angelin-Duclos C Cattoretti G Lin KI Calame K Commitment of B lymphocytes to a plasma cell fate is associated with Blimp-1 expression in vivo.J Immunol. 2000; 165: 5462-5471PubMed Google Scholar, 8Cattoretti G Angelin-Duclos C Shaknovich R Zhou H Wang D Alobeid B PRDM1/Blimp-1 is expressed in human B-lymphocytes committed to the plasma cell lineage.J Pathol. 2005; 206: 76-86Crossref PubMed Scopus (85) Google Scholar, 9Klein U Casola S Cattoretti G Shen Q Lia M Mo T Ludwig T Rajewsky K Dalla-Favera R Transcription factor IRF4 controls plasma cell differentiation and class-switch recombination.Nat Immunol. 2006; 7: 773-782Crossref PubMed Scopus (555) Google Scholar Both of these transcription factors are required for plasma cell differentiation.9Klein U Casola S Cattoretti G Shen Q Lia M Mo T Ludwig T Rajewsky K Dalla-Favera R Transcription factor IRF4 controls plasma cell differentiation and class-switch recombination.Nat Immunol. 2006; 7: 773-782Crossref PubMed Scopus (555) Google Scholar Microarray profiling demonstrates that PRDM1/Blimp-1 orchestrates plasma cell differentiation by repressing genetic programs associated with activated B cells and/or GC B cells, including those that control cell proliferation, and by activating genetic programs associated with plasma cell functions, including apoptosis.10Shaffer AL Lin KI Kuo TC Yu X Hurt EM Rosenwald A Giltnane JM Yang L Zhao H Calame K Staudt LM Blimp-1 orchestrates plasma cell differentiation by extinguishing the mature B cell gene expression program.Immunity. 2002; 17: 51-62Abstract Full Text Full Text PDF PubMed Scopus (791) Google Scholar Recent published data supported a role for interference of PRDM1 functions in the pathogenesis of human lymphomas. PRDM1 has been shown to be inactivated by a classic mechanism for tumor suppressor genes in large B-cell lymphoma diffuse (DLBCL), specifically those of the non-GCB type, strongly suggesting that inhibition of terminal B-cell differentiation may play a role in their pathogenesis.11Tam W Gomez M Chadburn A Lee JW Chan WC Knowles DM Mutational analysis of PRDM1 indicates a tumor-suppressor role in diffuse large B-cell lymphomas.Blood. 2006; 107: 4090-4100Crossref PubMed Scopus (198) Google Scholar, 12Pasqualucci L Compagno M Houldsworth J Monti S Grunn A Nandula SV Aster JC Murty VV Shipp MA Dalla-Favera R Inactivation of the PRDM1/BLIMP1 gene in diffuse large B cell lymphoma.J Exp Med. 2006; 203: 311-317Crossref PubMed Scopus (310) Google Scholar In addition, BCL-6-mediated transcription repression of PRDM1 causes blockade of terminal differentiation in GC-type DLBCL.13Parekh S Polo JM Shaknovich R Juszczynski P Lev P Ranuncolo SM Yin Y Klein U Cattoretti G Dalla Favera R Shipp MA Melnick A BCL6 programs lymphoma cells for survival and differentiation through distinct biochemical mechanisms.Blood. 2007; 110: 2067-2074Crossref PubMed Scopus (118) Google Scholar The neoplastic cells in classical Hodgkin lymphoma (HL), the Hodgkin/Reed-Sternberg (HRS) cells, resemble post-GC cells immunophenotypically and genetically, despite their putative origin from preapoptotic GC cells.14Staudt LM The molecular and cellular origins of Hodgkin's disease.J Exp Med. 2000; 191: 207-212Crossref PubMed Scopus (96) Google Scholar, 15Kuppers R Hansmann ML The Hodgkin and Reed/Sternberg cell.Int J Biochem Cell Biol. 2005; 37: 511-517Crossref PubMed Scopus (51) Google Scholar They frequently lack BCL-6 and consistently express IRF4.16Carbone A Gloghini A Aldinucci D Gattei V Dalla-Favera R Gaidano G Expression pattern of MUM1/IRF4 in the spectrum of pathology of Hodgkin's disease.Br J Haematol. 2002; 117: 366-372Crossref PubMed Scopus (87) Google Scholar, 17Carbone A Gloghini A Gaidano G Franceschi S Capello D Drexler HG Falini B Dalla-Favera R Expression status of BCL-6 and syndecan-1 identifies distinct histogenetic subtypes of Hodgkin's disease.Blood. 1998; 92: 2220-2228PubMed Google Scholar, 18Buettner M Greiner A Avramidou A Jack HM Niedobitek G Evidence of abortive plasma cell differentiation in Hodgkin and Reed-Sternberg cells of classical Hodgkin lymphoma.Hematol Oncol. 2005; 23: 127-132Crossref PubMed Scopus (50) Google Scholar They harbor somatic mutations in their immunoglobulin genes but show no evidence of ongoing somatic hypermutation.15Kuppers R Hansmann ML The Hodgkin and Reed/Sternberg cell.Int J Biochem Cell Biol. 2005; 37: 511-517Crossref PubMed Scopus (51) Google Scholar In addition, HL cell lines have a similar gene expression pattern as that of Epstein Barr Virus-transformed B cells and DLBCL cell lines showing features of in vitro-activated B cells.19Kuppers R Klein U Schwering I Distler V Brauninger A Cattoretti G Tu Y Stolovitzky GA Califano A Hansmann ML Dalla-Favera R Identification of Hodgkin and Reed-Sternberg cell-specific genes by gene expression profiling.J Clin Invest. 2003; 111: 529-537Crossref PubMed Scopus (217) Google Scholar Since HRS cells and non-GCB DLBCL appear to be in similar differentiation stages, we hypothesize a role of interference of PRDM1 functions in HRS cell pathogenesis. Sequence analysis has not identified inactivating mutation of PRDM1 in HL cell lines (unpublished data). Decrease in accumulation of PRDM1, however, may occur through quantitative changes in its synthesis or stability. One way by which PRDM1 synthesis can be altered is via regulation by miRNA. In fact, deregulation of target protein production by altered expression of miRNA is a well documented mechanism of oncogenesis.20Zhang W Dahlberg JE Tam W MicroRNAs in tumorigenesis: a primer.Am J Pathol. 2007; 171: 728-738Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar In this report, we provide experimental evidence that PRDM1 is a target for down-regulation by miRNAs in HRS cells. The GC-like DLBCL cell lines SUDHL6 and OCI-Ly1, the myeloma cell line U266, the primary effusion lymphoma (PEL) cell lines BC1, BC2, BC3, and BCBL1, and the HL cell lines L428, KMH2, and L1236, were cultured in RPMI medium 1640 with 10% heat-inactivated fetal calf serum (Invitrogen, Carlsbad, CA). 293T cells were maintained in Dulbecco's modified Eagle's medium with 10% heat-inactivated fetal bovine serum (Invitrogen). Formalin-fixed, paraffin-embedded archival tissue of classical HL cases were obtained according to the protocols approved by the Institutional Review Board. All samples were reviewed and classified according to the World Health Organization criteria. For Western blots and immunohistochemistry, a monoclonal antibody against human PRDM1 (ROS) was used. This antibody recognizes both PRDM1α and PRDM1β.21Garcia JF Roncador G Sanz AI Maestre L Lucas E Montes-Moreno S Fernandez Victoria R Martinez-Torrecuadrara JL Marafioti T Mason DY Piris MA PRDM1/BLIMP-1 expression in multiple B and T-cell lymphoma.Haematologica. 2006; 91: 467-474PubMed Google Scholar Immunoblotting and immunoperoxidase staining on paraffin tissue sections were performed as previously described.21Garcia JF Roncador G Sanz AI Maestre L Lucas E Montes-Moreno S Fernandez Victoria R Martinez-Torrecuadrara JL Marafioti T Mason DY Piris MA PRDM1/BLIMP-1 expression in multiple B and T-cell lymphoma.Haematologica. 2006; 91: 467-474PubMed Google Scholar Quantitation of PRDM1α expression in Western blots was done by densitometry and normalized with loading controls (lamin B or β-actin). Total RNA was extracted from cell lines and treated with RNase-free DNase I. For quantitative detection of PRDM1α mRNA, cDNA was synthesized from 1 μg of total RNA using random primers. Monoplex real-time PCR was conducted using ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). The PCR reaction was done using 50 ng of cDNA template according to the manufacturer's protocol using the following PCR conditions: 50°C for 2 minutes, 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, 58°C for 30 seconds, and 72°C for 1 minute. A standard curve, consisting of serially diluted U266 RNA, was included on each 96-well reaction plate for each run. Each cDNA template was assayed in duplicate, and each sample was run three independent times. Relative quantities of PRDM1α mRNA were calculated using the standard curve method, normalized, and expressed relative to U266 (set as one). For normalization, the 6 most stable reference genes identified using the freely distributed MicroSoft Excel application geNorm22Vandesompele J De Preter K Pattyn F Poppe B Van Roy N De Paepe A Speleman F Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.Genome Biol. 2002; 3 (RESEARCH0034. Epub 2002 Jun 18)Crossref PubMed Google Scholar among 11 candidate genes described previously23Lossos IS Czerwinski DK Wechser MA Levy R Optimization of quantitative real-time RT-PCR parameters for the study of lymphoid malignancies.Leukemia. 2003; 17: 789-795Crossref PubMed Scopus (132) Google Scholar were used. These six genes are: glyceraldehyde-3-phosphate dehydrogenase, protein kinase cGMP-dependent, type 1, hypoxanthine phosphoribosyltransferase 1, TATA box binding protein, large ribosomal phospoprotein PO, and β-glucoronidase. Primer and probe sequences for real-time detection of PRDM1α mRNA are as follows: Forward: 5′-TCCAGCACTGTGAGGTTTCA-3′; Reverse: 5′-TCAAACTCAGCCTCTGTCCA-3′; Probe: FAM-5′-ATGGACATGGAGGATGCGGATATG-3′-TAMRA. Real-time quantification of the endogenous control genes was performed using the Human Taqman predeveloped assays reagents endogenous controls (Applied Biosystems). For quantitative measurement of luciferase reporter mRNA in cell transfectants, cDNA was generated and real-time PCR was performed as described above using SYBR Green PCR Master Mix (Applied Biosystems). The relative Renilla luciferase mRNA levels (negative control transfectant set as 100) were calculated by the ΔΔCt method using firefly luciferase mRNA expression as normalization. Primer sequences are as follows: Renilla luciferase: Forward: 5′-AAGAGCGAAGAGGGCGAGAA-3′, Reverse: 5′-TGCGGACAATCTGGACGAC-3′; Firefly luciferase: Forward: 5′-CGTGCCAGAGTCTTTCGACA-3′, Reverse: 5′-ACAGGCGGTGCGATGAG-3′. Let-7a and miR-9 levels in cell lines were determined using TaqMan MicroRNA Assays (Applied Biosystems) following the protocols recommended by the manufacturer. Five ng of total RNA was used per 15 μl reaction during reverse transcription, and 1 μl of reverse transcription product was used for subsequent real-time PCR reactions. For each sample, three independent reverse transcription reactions were performed and each reaction was assayed in triplicate for real-time PCR. The levels of miRNAs were normalized with 5S RNA and the relative levels were calculated using the ΔΔCt method. Direct cloning of miRNAs was performed as previously described.24Pfeffer S Zavolan M Grasser FA Chien M Russo JJ Ju J John B Enright AJ Marks D Sander C Tuschl T Identification of virus-encoded microRNAs.Science. 2004; 304: 734-736Crossref PubMed Scopus (1323) Google Scholar Putative target sites for miRNA in the 3′ untranslated region (UTR) of human, mouse, rat, and chicken PRDM1 mRNA were identified using the MiRANDA Human miRNA Targets web site (http://cbio.mskcc.org/cgi-bin/mirnaviewer/mirnaviewer.pl) and the Targetscan (version 4.1) web site (http://genes.mit.edu/targetscan) based on the target prediction algorithms developed by John et al25John B Enright AJ Aravin A Tuschl T Sander C Marks DS Human MicroRNA targets.PLoS Biol. 2004; 2: e363Crossref PubMed Scopus (2973) Google Scholar and Lewis et al,26Lewis BP Shih IH Jones-Rhoades MW Bartel DP Burge CB Prediction of mammalian microRNA targets.Cell. 2003; 115: 787-798Abstract Full Text Full Text PDF PubMed Scopus (4250) Google Scholar respectively. The binding of chicken miR-9 to its target sequences is not depicted in either of these web sites, and was deduced based on the available chicken sequence database and with the assistance of the mFOLD program (version 3) available at the website http://mfold.bioinfo.rpi.edu/cgi-bin/rna-form1.cgi.27Zuker M Mfold web server for nucleic acid folding and hybridization prediction.Nucleic Acids Res. 2003; 31: 3406-3415Crossref PubMed Scopus (10412) Google Scholar The miR-155/BIC expression plasmid has been previously described.28Eis PS Tam W Sun L Chadburn A Li Z Gomez MF Lund E Dahlberg JE Accumulation of miR-155 and BIC RNA in human B cell lymphomas.Proc Natl Acad Sci USA. 2005; 102: 3627-3632Crossref PubMed Scopus (1206) Google Scholar To generate the miR-9-expressing plasmid pcDNA3.miR-9-1, a 515-bp DNA fragment encompassing pre-miR-9-1 was amplified by PCR using genomic DNA template with sense and antisense primers flanked at the 5′ end by EcoRI and BamHI sites, respectively. The sequences of the primers were 5′-ATATAGAATTCCCAAGCAGTGACCCAGA-3′ (sense) and 5′-TAATAGGATCCTTCCCTCCTACTCCCGCTGA-3′ (antisense). The PCR-generated fragment were digested with EcoRI and BamHI and subcloned into pcDNA3(+) (Invitrogen) between the EcoRI and BamHI sites. Construction of pSIC.PRDM1.3′UTR.538–2419 is as follows: A genomic fragment encompassing nucleotides 1 to 2419 of the PRDM1 3′UTR was PCR-amplified using primers flanked by XhoI and NotI sites, and cloned into pGEM-T-Easy (Promega, Madison, WI). The sequences of the primers were: 5′-CTCGAGGATTTTCAGAAAACACTTATTTTGTTTC-3′ (sense) and 5′-GCGGCCGCACATTTTGACAATTTGCACATAAATAAC-3′ (antisense). Sequence of the insert was confirmed by double-stranded sequencing. Plasmid DNA was isolated from recombinant clones and digested with XhoI and NotI. The insert was gel purified and cloned into psiCHECK-2 (Promega) downstream of the Renilla luciferase coding region between the XhoI and NotI sites. The resulting plasmid was then digested with XhoI and PshAI, followed by fill-in. The larger product was then gel purified and self-ligated to give the final product. The mutant reporter constructs were generated by using pSIC.PRDM1.3′UTR.538-2419 as a template and mutating the fifth and sixth positions (from the 3′end) of the putative miR-9 or let-7a binding sites using the GeneEditor in vitro Site-Directed Mutagenesis System (Promega). Mutations were generated in one or more of the three putative miR-9 binding sites, designated as Mut1, Mut2, Mut3, Mut(2 + 3), and Mut(1 + 2 + 3). The numbers 1 to 3 denote positions from the proximal to distal end. Sequence of the mutant report constructs thus obtained were confirmed by double-stranded sequencing. The mutagenic primers used (mutant nucleotides in italics) were as follows: miR-9.Mut1: 5′-CTTTTATTCTGCTAAGCCGTAAGATTACATGTTGG-3′; miR-9.Mut2: 5′-CTGAAGGTAAACGTAAGCATCACGTTGAC-3′; miR-9.Mut3: 5′-CAAAGTTAAAACTGACGTAAGTTACTGGCTTTTTAC-3′; and, let-7a: 5′-AGTTGTTCAACAACAGTTTGCTCATTGAGTGTGTCC-3′. 293T cells were transfected with 75 ng of miRNA expression plasmids (miR-9 or miR-155) or pcDNA3 (negative plasmid control) in 96-well plates using Effectene (Qiagen, Valencia, CA), or with 20 nmol/L of miRNA precursor molecules (let-7a or negative miRNA control) purchased from Ambion (Austin, TX) in 24-well plates using Lipofectin (Invitrogen). For experiments evaluating the cooperative effects of miR-9 and let-7a, 50 nmol/L of either miRNA was transfected. These plasmids and miRNA analogs were co-transfected with 25 ng of pSIC.PRDM1.3′UTR.538–2419 (wild type or mutants), respectively. Twenty-four hours after transfection, Renilla and firefly luciferase activities were determined using the Dual-Glo Luciferase Assay System (Promega). The Renilla luciferase activities were normalized by firefly luciferase activities, which served as internal controls. The normalized values were compared with that of the negative controls to determine percentage change. The mean values (±SE) from three or four independent experiments are shown. Optimized nucleofection protocols generated by Amaxa (Cologne, Germany) were followed for the transfection of U266 (Nucleofector Kit C, Program X-005) and L428 (Nucleofector Kit L, Program X-001) with miRNA precursors (Ambion) or anti-miRNA inhibitors (Ambion), respectively. A total of 100 nmol/L was used for all transfections. For co-transfections of anti-miR-9 and anti-let-7a inhibitors, 50 nmol/L each was added. Cells were collected at 24 to 48 hours after electroporation and subject to Western blot analysis for determination of PRDM1 expression. These transfection experiments were repeated four independent times. P values were calculated by Student's t-test using the StatView software (SAS Institute Inc., Cary, NC). As a first step to identify miRNAs that may regulate PRDM1 expression, miRNA profiles for HL cell lines L428, KMH2, and L1236 generated by direct cloning29Landgraf P Rusu M Sheridan R Sewer A Iovino N Aravin A Pfeffer S Rice A Kamphorst AO Landthaler M Lin C Socci ND Hermida L Fulci V Chiaretti S Foa R Schliwka J Fuchs U Novosel A Muller RU Schermer B Bissels U Inman J Phan Q Chien M Weir DB Choksi R De Vita G Frezzetti D Trompeter HI Hornung V Teng G Hartmann G Palkovits M Di Lauro R Wernet P Macino G Rogler CE Nagle JW Ju J Papavasiliou FN Benzing T Lichter P Tam W Brownstein MJ Bosio A Borkhardt A Russo JJ Sander C Zavolan M Tuschl T A mammalian microRNA expression atlas based on small RNA library sequencing.Cell. 2007; 129: 1401-1414Abstract Full Text Full Text PDF PubMed Scopus (3045) Google Scholar were examined (Figure 1). Based on these expression profiles, we selected for further analysis two miRNAs, miR-9 and let-7a, with high potential to functionally interact with PRDM1 mRNA. Both these miRNAs are among the 10 most abundant miRNAs in HL cell lines and constitute on average about 3.5% and 1.9% of the total miRNA population, respectively. These expression levels rank close to miR-155, a highly expressed miRNA in HRS cells.30Kluiver J Poppema S de Jong D Blokzijl T Harms G Jacobs S Kroesen BJ van den Berg A BIC and miR-155 are highly expressed in Hodgkin, primary mediastinal and diffuse large B cell lymphomas.J Pathol. 2005; 207: 243-249Crossref PubMed Scopus (583) Google Scholar, 31Tam W Dahlberg JE miR-155/BIC as an oncogenic microRNA.Genes Chromosomes Cancer. 2006; 45: 211-212Crossref PubMed Scopus (104) Google Scholar In addition, putative binding sites for these miRNAs, including three for miR-9 and one for let-7, are predicted in the PRDM1 3′UTR independently by two computer algorithms: miRanda and Targetscan (release 4.1, January 2008).25John B Enright AJ Aravin A Tuschl T Sander C Marks DS Human MicroRNA targets.PLoS Biol. 2004; 2: e363Crossref PubMed Scopus (2973) Google Scholar, 32Lewis BP Burge CB Bartel DP Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets.Cell. 2005; 120: 15-20Abstract Full Text Full Text PDF PubMed Scopus (9936) Google Scholar, 33Grimson A Farh KK Johnston WK Garrett-Engele P Lim LP Bartel DP MicroRNA targeting specificity in mammals: determinants beyond seed pairing.Mol Cell. 2007; 27: 91-105Abstract Full Text Full Text PDF PubMed Scopus (3049) Google Scholar The pairing between these miRNAs and their target sequences are evolutionarily conserved, with absolute conservation between the 6bp “seed” region32Lewis BP Burge CB Bartel DP Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets.Cell. 2005; 120: 15-20Abstract Full Text Full Text PDF PubMed Scopus (9936) Google Scholar at the 5′ end of the miRNA and the complementary sequence of the target site (Figure 2). Conserved binding sites for two other miRNAs, miR-125/351 and miR-365, are also predicted in the PRDM1 3′UTR by both miRanda and Targetscan; however, these miRNAs are present at very low amount in HL cell lines based on miRNA cloning (<0.5% of the total miRNA population) and therefore not selected for further analysis.Figure 2PRDM1 3′UTR harbors putative target sites for miR-9 and let-7a. A: A schematic representation of the PRDM1 3′ UTR is shown. The positions of the putative miR-9 and let-7a sites are indicated by arrows, and other conserved miRNA binding sites predicted by TargetScan (v.4) only or by both TargetScan and MiRANDA are marked by open and filled triangles, respectively. The portion of the PRDM1 3′ UTR that is cloned and used for the luciferase reporter assays is marked in bold line. B and C: Complementarity between miR-9 or let-7a (top strand) and its conserved putative binding sites (bottom strand) in the PRDM1 3′ UTR is shown for different species. A Watson-Crick bp is indicated by a vertical line, and the U:G wobble base is marked by double dots. The highly conserved 6-bp “seed” pairing is highlighted. The numbers indicate the locations of the putative binding sites in the PRDM1 3′ UTRs downstream from the PRDM1 stop codon.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Luciferase reporter assays were performed to determine whether miR-9 and let-7a can target the 3′UTR of PRDM1 mRNA. The reporter plasmid, psiC.PRDM1.3′UTR.538–2419, contains the majority of the PRDM1 3′UTR that harbors all of the putative binding sites for the two miRNAs (Figure 2A). This plasmid was cotransfected into 293T cells with miR-9, let-7a, miR-155, or negative miR controls. No mir-155 binding sites were predicted in the PRDM1 3′UTR. MiR-9 and let-7a decreased normalized Renilla luciferase activities to 39.2% ± 4.6%, and 41.7% ± 3.8% of negative controls, respectively, whereas cotransfection with miR-155 showed no significant difference (Figure 3A). Reduction in luciferase activities by miR-9 and let-7a is mediated mainly by translation repression. No significant difference in Renilla luciferase mRNA levels was detected among the negative control, miR-9 and let-7a transfectants after normalization, although miR-9 transfectants may tend to show a slight reduction (Figure 3B). To demonstrate that both miRNAs interact specifically with their predicted target sequences, additional reporter constructs harboring mutations in the “seed pairing” sequences of the putative binding sites were generated using site-directed mutagenesis. These mutated reporter constructs were transfected into 293T cells with the corresponding miRNAs as described above and luciferase reporter assays were measured. Mutations in the predicted target sites for each of these miRNAs relieve repression of luciferase activities (Figure 3A). These results indicate that miR-9 and let-7 can repress translation by direct and specific interaction with the PRDM1 3′UTR. Interestingly for miR-9, the extent of de-repression depends on which and how many miR-9 binding sites are mutated. Mutations in the most proximal site (Mut 1) resulted in partial relief in repression (77.0% ± 2.5% of negative control), whereas mutations in each of the other two miR-9 binding sites (Mut 2 and Mut 3) do not have significant effects compared with wild type (48.6% ± 2.4% and 45.2% ± 2.6%). Mutations in the middle and distal sites (Mut[2 + 3]) resulted in a slight de-repression (59.1% ± 4.7% of negative control), whereas mutations in all three miR-9 binding sites (Mut[1 + 2 + 3]) led to a complete de-repression (117.5% ± 9.9%). These results suggest a differential, as well as cooperative, repression effect for the three miR-9 target sites in the PRDM1 3′ UTR. We tested whether interaction of both miR-9 and let-7a with the PRDM1 3′UTR may increase the repression effects relative to either miRNA alone by cotransfecting psiC.PRDM1.3′UTR.538–2409 with miR-9 and let-7a. Cotransfection of miR-9 and let-7a (each at 50 nmol/L) reduced luciferase activity to 19.7% ± 1.5% of negative control, significantly different from the effects of miR-9 or let-7a alone (P < 0.05 and P < 0.001, respectively) (Figure 3B). This increase in repression is likely due to additive effects of miR-9 and let-7a, because similar repression was obtained when miR-9 concentration was increased to 100 nmol/L. Let-7a at 50 nmol/L or 100 nmol/L shows no significant difference in repression, suggesting that it has already reached saturation at these concentrations. If miR-9 and let-7a functionally interact with endogenous PRDM1 mRNA to down-regulate PRDM1 protein expression in HRS cells, we should expect association of high levels of miR-9 and let-7a with relatively lower levels of PRDM1 in these cells. For comparisons, we selected cell lines that exhibit plasmablastic/plasmacytic differentiation such as PEL and myeloma cell lines, and cell lines that correspond to the GC differentiation stage such as the GC-DLBCL cell lines. Real-time RT-PCR analysis confirmed the miRNA cloning data and
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