PRDM1bβ expression in diffuse large B-cell lymphoma.

Just Accepted by Leukemia & Lymphoma

Methylation Contributes to the Imbalance of PRDM1α/PRDM1β Expression in Diffuse Large B-cell Lymphoma Yi-Wen Zhang, Jie Zhang, Jun Li, Jun-Feng Zhu, Yan-Li Yang, Li-Li Zhou, Zhong-Li Hu, Feng Zhang doi: 10.3109/10428194.2014.994181 Leuk Lymphoma Downloaded from informahealthcare.com by Yale Dermatologic Surgery on 12/30/14 For personal use only.

Abstract The positive regulatory domain 1 (PRDM1) exists as two isoforms: PRDM1a and PRDM1b, the former is frequently inactivated, while the latter is overexpressed in a subset of diffuse large B-cell lymphomas (DLBCL). To investigate the possible epigenetic alteration of PRDM1a and PRDM1b expression, the methylation of these two isoforms promoters was assessed in B lymphoma cell lines and DLBCL samples. Hypomethylation of PRDM1β CpG islands was preferentially detected in lymphoma cells. However, both high and low methylation of PRDM1α CpG islands were simultaneously observed in DLBCL cases compared with the moderate methylation of non-tumor cases. CpG 16–21-specific high methylation was correlated with low expression of PRDM1α in PRDM1b-positive DLBCL samples. Three increased and one decreased miRNAs were significant difference between DLBCL and non-tumor reactive hyperplasia cases. Thus, our results indicated that aberrant methylation silencing of PRDM1α and hypomethylation activation of PRDM1β were frequent events in DLBCL.

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Methylation Contributes to the Imbalance of PRDM1α/PRDM1β Expression in Diffuse Large B-cell Lymphoma

Yi-Wen Zhang1,2,#, Jie Zhang2,#, Jun Li1, Jun-Feng Zhu1, Yan-Li Yang1, Li-Li Zhou1, Zhong-Li Hu1, Feng Zhang1

1

Department of Hematology, The First Affiliated Hospital of Bengbu Medical College, Bengbu, Anhui Province, China,

2

Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Key Laboratory of Thrombosis

Leuk Lymphoma Downloaded from informahealthcare.com by Yale Dermatologic Surgery on 12/30/14 For personal use only.

and Hemostasis of Ministry of Health, Suzhou, Jiangsu Province, China

# These authors contributed equally to this manuscript Correspondence author: Yi-Wen Zhang & Feng Zhang, Department of Hematology, The First Affiliated Hospital of Bengbu Medical College, 287 Changhuai Road, Bengbu 233004, China. Tel: +86-552-3086384. Fax: +86-552-3070260. E-mail: [email protected] and [email protected], and Jiangsu Institute of Hematology, Hematology, 708 Renming, Suzhou, 215006 China Email: [email protected]

Short title: Aberrant methylation of PRDM1 promoter Abstract The positive regulatory domain 1 (PRDM1) exists as two isoforms: PRDM1α and PRDM1β, the former is frequently inactivated, while the latter is overexpressed in a subset of diffuse large B-cell lymphomas (DLBCL). To investigate the possible epigenetic alteration of PRDM1α and PRDM1β expression, the methylation of these two isoforms promoters was assessed in B lymphoma cell lines and DLBCL samples. Hypomethylation of PRDM1β CpG islands was preferentially detected in lymphoma cells. However, both high and low methylation of PRDM1α CpG islands were simultaneously observed in DLBCL cases compared with the moderate methylation of non-tumor cases. CpG 16–21-specific high methylation was correlated with low expression of PRDM1α in PRDM1β-positive DLBCL samples. Three increased and one decreased miRNAs were significant difference between DLBCL and non-tumor reactive hyperplasia cases. Thus, our results indicated that aberrant methylation silencing of PRDM1α and hypomethylation activation of PRDM1β were frequent events in DLBCL.

Keywords: PRDM1, isoform, epigenetic, DNA methylation, diffuse large B-cell lymphoma

1

Introduction Human positive regulatory domain 1 (PRDM1) is located on chromosome 6q21, belonging to the PRDM family of transcription repressors, simultaneously contains Krüppel-type zinc fingers and the PR domain. The PR domain is a subclass of the SET domain that methylates histone H3 on lysine 9 (H3K9). In human cancers, the histone methyltransferase (HMT) activity is reduced by mutations in


the PR domain [1]. Several PRDM family members, including PRDM1, share the common feature of expressing two products differing in the presence or absence of the PR domain. In cancer cells, the PR-plus product is disrupted or downexpressed, whereas the PR-minus product is present or overexpressed. These two proteins are generally kept in a special balance in an unusual yin-yang fashion [2]. Once the balance relationship is broken, a series of either genetic or epigenetic events appears to be critical for oncogenesis. PRDM1 exists as two isoforms: α and β. The former isoform was found to be inactivated in the non-germinal center B (GCB) subtype of DLBCL and is considered as a potential tumor suppressor gene in these lymphomas [3-5]. On the other hand, the latter isoform is transcribed from an alternate promoter located in intron 3 and part of exon 4 of the full-length PRDM1α. PRDM1β lacks the first 101 amino acids at the N-terminal of PRDM1α and bears part of the PR domain. An intact PR domain is required for tumor suppressing functions. Therefore, PRDM1β has a significantly impaired transcription repressor function on multiple target genes [6]. It has been verified that the ratio of PRDM1α/PRDM1β in normal plasma cells was remarkably higher than that of multiple myeloma patients [7], which is consistent with no significance in the expression of PRDM1α, and otherwise significant difference of PRDM1β in other groups. Recently, mounting evidence for PRDM1α as a tumor suppressor gene in hematological malignancies [8-14] and silencing of PRDM1 gene in non-GCB DLBCL [3,4,11] has been frequently reported. The PRDM1β isoform was overexpressed in multiple myelomas [7], non-GCB DLBCL [15], and in some T-cell lymphomas, but 2

not in non-tumor cells or tissues [16]. Our previous study also demonstrated that both PRDM1α and PRDM1β were expressed in microdissected lymphoma tissue cells only in the non-GCB cell-like subtype of diffuse large B-cell lymphoma (DLBCL) and T-cell lymphoma [15,16]. PRDM1β gene expression was correlated with a short patient survival time. However, their regulation mechanism remains to be investigated. Although PRDM1 mutations occur in about 25% of non-GCB DLBCL, the


majority of the other cases of this subtype lack PRDM1 protein, suggesting that additional mechanisms may inhibit PRDM1 translation or stability [4]. DNA cytosine methylation in CpG dinucleotides is an important epigenetic event that is critical for the control of gene expression. The methylation in CpG-rich promoter regions results in the transcriptional repression of the corresponding genes, whereas demethylation is required for stable expression. Promoter DNA methylation is a common mechanism for inactivating tumor suppressor genes in tumorigenesis. Our previous study demonstrated that the upregulation of the PRDM1β isoform was associated with the hypomethylation of the PRDM1β specific promoter in non-GCB DLBCL with aggressive behavior [17]. However, the methylation pattern of PRDM1α and its correlation with PRDM1β should be further investigated. Recently, it was demonstrated that PRDM1 is also a target for miRNA-mediated downregulation by miR-9 and let-7a in Reed-Sternberg/Hodgkin cells of Hodgkin lymphoma. In addition, high levels of miR-9 and let-7a in HL cell lines correlated with low levels of PRDM1 [8]. Moreover, it was also demonstrated that the miRNA let-7 family mediates the translational downregulation of PRDM1 in non-GCB DLBCL [18], suggesting that miRNAs may be involved in the regulation of the ratio PRDM1α/PRDM1β in DLBCL. In the present study, we assessed the methylation status of PRDM1α and PRDM1β in B lymphoma cell lines and primary DLBCL samples. Our results indicated that aberrant methylation silencing of PRDM1α and hypomethylation activation of PRDM1β were frequent events in DLBCL. We also 3

identified the differential expression of some miRNAs regulated by DNA methylation. The aberrant patterns of methylation might contribute to the imbalance of the PRDM1α to PRDM1β ratio. Materials and Methods CELL LINES AND REAGENTS Human B lymphoma cell lines Namalwa and SU-DHL-4 (American Type Culture Collection,


Manassas, VA, USA) were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco/BRL, Grand Island, NY, USA) in 5% CO2-95% air humidified atmosphere at 37°C. Cells from the human embryonic kidney 293T (HEK 293T) cell line and umbilical vein cell line EA.hy926 (American Type Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium under the same conditions. Human normal B-cells were purified by flow cytometric sorting using anti-CD19 from peripheral blood of healthy volunteers. DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-aza) was obtained commercially (Sigma-Aldrich Co Ltd, St. Louis, USA). PATIENTS Twenty-six de novo DLBCL patients of non-GCB subtype with available tumor specimen at diagnosis were included in this study. The histological diagnoses were established according to World Health Organization classifications and the non-GCB phenotype was defined by immunohistochemical staining of CD10, BCL-6, and IRF4/MUM1. All the participants were all informed and written consent form. For this study, further scientific research and ethical approval was obtained from the Ethics Committee in the Bengbu Medical College (2013 No.11). TISSUE SAMPLES Tumor biopsies at time of diagnosis were immediately cut into two parts: one part was fixed in formaldehyde and further processed for paraffin embedding, and the other was snap-frozen and stored at −80°C. 4

SEMI-QUANTITATIVE REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION (RT-PCR) Total RNA was extracted from frozen tissue sections or cultured cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized using MMLV reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. Semi-quantitative RT-PCR


was performed using the PRDM1α and PRDM1β primers, and GAPDH was employed for normalization (Supplement Table 1). The relative positions of the primers were indicated in Supplement Figure 1. BISULFITE SEQUENCING PCR (BSP) The CpG islands of the PRDM1α and PRDM1β promoters were identified using the Methyl Primer Express Software v1.0 (Applied Biosystems, Foster City, CA, USA) from 5000 bp upstream of the transcriptional start point. DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega Corporation, Madison, WI, USA) according to the manufacturer’s instructions. After treatment with the EZ DNA Methylation Gold Kit (Zymo Research, Orange, CA, USA), bisulfite PCR was performed to assess the methylation of the CpG islands of the PRDM1α and PRDM1β promoters, using the PRDM1α and PRDM1β BSP primers (Supplement Table 1). The amplified fragments were cloned into the pGEM-T Easy vector using the TOPO-TA cloning kit (Invitrogen), sequenced by the BigDye terminator kit on an ABI 3100 automated sequencer (Applied Biosystems), and at least eight clones were sequenced per sample. The methylation status of each CpG site of each clone is indicated. LUCIFERASE REPORTER ASSAY Three and four constructs of different size covering the region upstream of the PRDM1α and PRDM1β promoters were created, respectively. Nested deletions of the PRDM1α and PRDM1β promoter regions were carried out by PCR amplification, using the plasmids pα (786–1382) and pβ (587–1066) 5

as the templates, and the sequences of the products were confirmed by direct sequencing. The primers for nested deletions are listed in Supplement Table 1. The restriction endonuclease KpnI site was inserted in the upstream primer, while the HindIII site was in the downstream primer. All the amplified products were subcloned into the pGEM-T Easy vector, excised by KpnI and HindIII (TaKaRa Bio Inc., Japan), and inserted into the KpnI and HindIII sites upstream of the promoter fragments of the


pGL4.15 Luciferase reporter plasmids (Promega). EA.hy926 and HEK 293T cells (1.5 × 105 cells in 12-well plates) were transiently co-transfected with 2 μg of the reporter gene construct, as well as with 2 ng of SV40 plasmid to normalize the transfection efficiency into each well by using the CaPO4-DNA co-precipitation method. The pGL4.15-Basic plasmid without the insert was used as the negative control. Thirty-six hours after transfection, cells were lysed with passive lysis buffer (Dual Luciferase Reporter Assay System, Promega). Luciferase activities were measured by Lumat LB9507 luminometer (EG&G Berthold, Bad Wildbad, Germany). IN VITRO METHYLATION OF PRDM1α and PRDM1β PROMOTER The promoter-luciferase reporter construct (5 μg) described above was treated with or without (mock) SssI (CpG) methyltransferase (4 units) (New England BioLabs, Ontario, Canada) plus S-adenosylmethionine (640 μM) in the manufacturer’s recommended buffer (5 μL) at 37°C for 4 h, followed by incubation at 65°C for 20 min to inactivate the enzymes. Following plasmid purification, the methylation status was confirmed by restriction digestion with the methylation-sensitive restriction endonuclease HpaII, which recognizes only the unmethylated cytosine of the CpG regions. The plasmid construct could not be separated when cytosine was artificially methylated by SssI methyltransferase. The methylated and mock-methylated plasmids were then transfected into HEK 293T cells. The luciferase activity assay was performed as described above.

6

MASSARRAY QUANTITATIVE DNA METHYLATION ANALYSIS (MAQMA) MAQMA is a quantitative assay that utilizes matrix-assisted laser desorption/ionization time of flight mass spectrometry and base-specific cleavage to analyze DNA methylation patterns in sodium-bisulfite converted DNA. Sodium-bisulfite conversions were accomplished using the EZ DNA methylation kit (Zymo Research, Orange, CA). The methylation PCR assay of the PRDM1α


promoter can be analyzed directly using PRDM1α-EPI primers by the MassARRAY system and EpiTYPER software (Sequenom, San Diego, CA). However, multiplex-nested methylation PCR of PRDM1β was used. The nested method initially amplifies bisulfite-modified DNA using FLANK PCR primers; then, the 1:100-diluted FLANK PCR products are employed as the INSIDE PCR templates for CpG island methylation of the PRDM1β promoter using INSIDE PCR primers. The primers used for MAQMA analysis are shown in Supplement Table 1. MICRORNA MICROARRAY ANALYSIS Namalwa cells were incubated in the absence or presence of 5-aza (1 μM). Half of the medium was changed every other day to maintain a cell density of 0.5–1 × 106/mL. After incubation for three days, the total RNA was harvested using TRIzol (Invitrogen) and the miRNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. A miRCURY™ LNA Array (v.18.0) (Exiqon, Vedbaek, Denmark) was performed by KangChen Bio-technology in Shanghai. RNA samples were labeled using the miRCURY™ Hy3™/Hy5™ Power labeling kit and hybridized on the microarray. Following the washing steps, the slides were scanned using the Axon GenePix 4000B microarray scanner. The scanned images were then imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction. The replicated miRNAs were averaged, and miRNAs exhibiting intensities ≥50 in all samples were chosen for calculating the normalization factor. The data were normalized using the median normalization. After normalization, significantly differentially expressed 7

miRNAs were identified through Volcano Plot filtering. Finally, hierarchical clustering was performed to show distinguishable miRNA expression profiling among samples. The analysis dataset had been submitted to Gene Expression Omnibus (GEO) and assigned accession number GSE53666 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE53666). MICRORNA REAL TIME RT-PCR ARRAY


Up- and downregulated miRNAs were quantitated by real time RT-PCR to confirm the reliability of the miRNA array assay. Briefly, ten de novo B-cell lymphoma and reactive hyperplasia samples were included in this assay. After total RNA was extracted and cDNA reverse transcripted, 2 μL of cDNA was mixed with 5 μL of 2× SYBR green master mix (Applied Biosystems Inc., Foster City, USA), 0.5 μL of each of 10 μM forward and reverse gene-specific primers (Supplement Table 3), and water to a final volume of 10 μL. The reactions were amplified at 95°C for 10 min followed by 40 cycles at 95°C for 10 s and 60°C for 60 s. U6 small nuclear RNA (U6) served as the endogenous control. The relative amount of each miRNA to U6 was described using the formula 2−ΔCt, where ΔCt = (CtmiRNA − CtU6). Each sample was run in triplicate. STATISTICAL ANALYSIS All experiments were performed in triplicate and the results were expressed as the mean ± standard deviation (SD). The variance between the groups was measured by two-tailed t-test. P < 0.05 was considered to be significant. All statistical analyses were performed with the SPSS 18.0 software. Results/Discussion THE RATIO OF PRDM1α/PRDM1β SIGNIFICANTLY DECREASED IN NAMALWA LYMPHOMA CELLS COMPARED TO SU-DHL-4 AND COULD BE RESTORED BY 5-AZA The relative expression levels of PRDM1α and PRDM1β were assessed by semi-quantitative RT-PCR. As shown in Figure 1A and 1B, PRDM1α was expressed at high levels in non-tumor cells (HEK 293T 8

and normal B), and no significant difference was found between the two B lymphoma cells (Namalwa and SU-DHL-4). However, Namalwa cells expressed a relatively higher level of PRDM1β than SU-DHL-4 cells, while no PRDM1β expression was detected in HEK 293T cells and human normal B-cells. We further examined the ratio of the PRDM1α and PRDM1β levels in the two B lymphoma cells, in which the lowest ratio was found in Namalwa cells (Figure 1C). Moreover, the ratio could be


restored when SU-DHL-4 cells were treated with the methyltransferase inhibitor 5-aza (Figure 1D and 1E), with cell survival remaining at about 70% (Figure 1F). These results indicated that DNA methylation in DLBCL may be involved in silencing the expression of PRDM1α and activating the expression of PRDM1β isoform. HYPOMETHYLATION OF BOTH PRDM1α AND PRDM1β PROMOTERS IN NAMALWA CELLS WHILE HYPERMETHYLATION IN SU-DHL-4 We next used BSP to assess the methylation of the CpG islands of the PRDM1α and PRDM1β promoters in the two B-cell lymphoma and normal cell lines. One CpG island was identified at 986–1234 bp and 826–1025 bp (1001 bp is the transcription start site, TSS) of the PRDM1α and PRDM1β promoters, respectively (Figure 2A and 3A). The results of BSP revealed that Namalwa cells showed hypomethylation (α, 1.4%; β, 7.8%), SU-DHL-4 cells showed hypermethylation (α, 67.8%; β, 39.1%), and HEK 293T cells showed trace or null methylation of PRDM1α but almost full methylation of PRDM1β (α, 0%; β, 93.8%). Another non-tumor cell line, EA.hy926, presented hypermethylation of either PRDM1α or PRDM1β genes (α, 53.4%; β, 85.9%) (Figure 2B, 2C and Figure 3B, 3C). Correlation analysis of mRNA expression suggested a significant association of methylation with lower expression of PRDM1α and higher expression of PRDM1β in B lymphoma cells than that in non-tumor cells. The involvement of methylation alteration has also been reported for other PRDM family genes like PRDM2 and PRDM5 in solid tumors [19,20]. 9

METHYLATION OF THE PRDM1α AND PRDM1β PROMOTER FUNCTIONALLY REPRESSED GENES TRANSCRIPITION Using the luciferase assay, the specific region of the CpG islands responsible for the PRDM1α and PRDM1β promoter activity was evaluated. One plasmid of the whole CpG islands and three and four constructs covering different regions upstream the TSS of the PRDM1α and PRDM1β promoters,


respectively, were created. Promoter activity was detected in all the cell lines tested, regardless of the gene expression levels. For PRDM1β, most of the luciferase activity resided in the construct that contained the sequence 587–999 bp, and the value was almost the same as the construct covering the whole CpG island, which was approximately 5-fold higher than that of the other two fragments. This suggested that the sequence 587–999 bp, was the region responsible for the PRDM1β expression (Figure 4B). It is worth noting that most of the promoter activity of PRDM1α resided in the truncated sequence 786–1039 bp rather than in the whole CpG island, which suggested that the truncated sequence 786–1039 bp, might play an important role in PRDM1α expression (Figure 4A). In addition, the sequence 1024–1234 bp probably contains transcription repressor binding sites necessary for a proper regulation of the expression of PRDM1α in DLBCL. To elucidate whether the transcription activity of the PRDM1α and PRDM1β isoforms was regulated in a CpG methylation-dependent manner, in vitro methylation was performed on the plasmid bearing the sequences 786–1039 bp and 587–999 bp, which presented the most significant promoter activity. Compared to that of the unmethylated plasmid, the relative luciferase activity of the SssI-methylated plasmid was significantly repressed approximately 11- and 6-fold, respectively, when compared with the unmethylated version (Figure 4C). Thus, the transcription activity of the PRDM1α and PRDM1β promoter regions was regulated in a CpG methylation-dependent manner, and the ratio of the PRDM1α/PRDM1β alteration is associated with the methylation of these regions. 10

ABERRANT CpG-SPECIFIC METHYLATION OF THE PRDM1α PROMOTER AND HYPOMETHYLATION OF PRDM1β IN DLBCL Our previous studies indicated that both PRDM1α and PRDM1β transcripts were expressed only in the non-GCB subtype of DLBCL, and that PRDM1β expression was an independent adverse prognostic factor for event-free survival (EFS) and overall survival (OS). No correlation was found between


PRDM1α gene expression and survival [15]. Accordingly, the non-GCB subtype could be divided into two sub-subtypes: PRDM1β-positive or -negative B-cell lymphoma. In the present study, we investigated the methylation level of the PRDM1α promoter on the basis of whether PRDM1β expresses. We recently also verified that the presence of the PRDM1β isoform was associated with the loss of epigenetic silencing and hypomethylation in non-GCB DLBCL [17]. To our knowledge, this is the first report providing evidence that isoforms of a tumor suppressor gene have distinct methylation profiles. The gene is hypomethylated and transcriptionally active in tumor cells, while it is hypermethylated and transcriptionally inactive in the normal counterparts. However, whether the expression of PRDM1α is related to its promoter methylation in DLBCL remains unclear. Here, we continued to use the MAQMA platform to perform methylation analyses to cross-validate our previous findings and extended our analysis to all the available non-GCB DLBCL cases. The region for analysis included the CpG dinucleotides upstream of the TSS (1001 bp), including 31 CpG dinucleotides in the 923–1220 bp region of the PRDM1α promoter and 11 CpG dinucleotides in the 872–1134 bp of the PRDM1β promoter. As previously described, since PRDM1β was expressed in the lymphoma cells of DLBCL with non-GCB subtype, the methylation profile was assessed in tissue samples of 26 non-GCB DLBCL cases and 14 reactive hyperplasia cases. Consistent with our previous study, the CpG sites of the PRDM1β isoform were densely methylated in reactive hyperplasia, while sparsely methylated in non-GCB DLBCL. Compared with PRDM1β-positive DLBCL, methylation was more 11

frequently observed in PRDM1β-negative DLBCL (Figure 5B). However, a significant difference was observed only between PRDM1β-positive DLBCL and reactive hyperplasia samples (P < 0.0001, Figure 5D). In PRDM1α, the methylation profile was variable. Compared with PRDM1β-positive DLBCL, methylation was more frequently observed in PRDM1β-negative DLBCL. However, the reactive hyperplasia cases had moderate methylation rather than dense hypermethylation of the


PRDM1β promoter (Figure 5A). There was significant difference between either PRDM1β-positive DLBCL or reactive hyperplasia samples and PRDM1β-negative DLBCL (P < 0.0001, Figure 5C), but there was no difference between PRDM1β-positive DLBCL and reactive hyperplasia cases (P > 0.05, Figure 5C). To determine which site was the specific CpG dinucleotide of the PRDM1α CpG island in non-GCB DLBCL and reactive hyperplasia samples, 31 CpG dinucleotides were analyzed. Overall, we observed significantly higher methylation levels of the CpG 7 and CpG 16–21 sites in non-GCB DLBCL samples than those in non-tumor reactive hyperplasia samples. The mean methylation levels in CpG 7 and CpG 16–21 were 19.5% and 27%, respectively, compared with the 3% of the remaining grouped CpGs (P < 0.0001, Figure 5E). Difference analysis was performed to evaluate the relationship between CpG 7 and CpG 16–21 and PRDM1β expression within the same samples. There was significant difference between the expression of PRDM1β in non-GCB DLBCL and the methylation in CpG 16–21 (Figure 5G), but not in CpG 7 (Figure 5F). Therefore, the methylation heterogeneity of CpG 16–21 was cell-specific or DLBCL subtype-specific. In the present study, the hypomethylation of the PRDM1α promoter is correlated with PRDM1β-positive DLBCL in accordance with the relatively higher expression of PRDM1α, while hypermethylation is associated with PRDM1β-negative DLBCL, consistent with lower PRDM1α levels.

12

Recently, two groups had also reported on the distinct methylation status of the PRDM1α promoter in different classes of lymphoma. Nie et al. proposed the absence of hypermethylation of the PRDM1α promoter in GCB DLBCL patients and cells [18]. However, another group demonstrated that the PRDM1α promoter CpG island is hypermethylated in natural killer (NK) cell lines and natural killer cell lymphoma (NKCL) cases (12 out of 17, 71%). PRDM1 is also a tumor suppressor gene in NKCL


[9,21]. In our present study, the methylation heterogeneity of the PRDM1α promoter CpG island was revealed also in a sub-subtype of non-GCB DLBCL. These data indicated that tumorigenesis depends on multiple factors, including the imbalance between tumor suppressor genes and oncogenes. Our previous studies and published literature confirmed that the overexpression of the PRDM1β isoform is associated with advanced Ann Arbor stage and high risk International Prognostic Index (IPI) in T-cell lymphoma, with shorter survival in both DLBCL and T-cell lymphoma patients. In both B- and T-cell lymphomas, PRDM1β expression is also associated with in vitro resistance to chemotherapeutic agents. Here, our results further explained that aberrant methylation contributing to the imbalance of PRDM1α/PRDM1β might be associated with the lymphomagenesis. METHYLATION IS INVOLVED IN THE REGULATION OF THE EXPRESSION LEVEL OF MICRORNAS IN DLBCL To identify whether methylation was involved in regulating the expression level of miRNAs in DLBCL, we performed miRNA microarray analysis in the Namalwa cell line that was considered as non-GCB DLBCL after treatment with 5-aza. The result of the hierarchical clustering showed distinctive miRNA expression patterns in Namalwa cells treated with or without 5-aza (Figure 6A). Overall, the expression of 58 miRNAs was changed at least 1.5-fold, 27 of which were upregulated and 31 were downregulated (Supplement Table 2). We then examined the differential expression level of the top three up- and down-regulated miRNAs from 10 non-GCB DLBCL patients and 10 reactive 13

hyperplasia cases using real time RT-PCR. All the three elevated miRNAs (i.e., miR-4754, miR-361-5p and miR-320c) displayed significant difference between the two groups, while among the top three decreased miRNAs (i.e., miR-92b-3p, miR-3175 and miR-933) only miR-92b-3p was significantly different between DLBCL and non-tumor reactive hyperplasia cases. However, it will be necessary in the future to conduct larger studies.


Of note, miR-320c may be a potential and promising target for the pathogenesis and treatment of tumors. The expression level of miR-320c is distinct in various types of tumors. It is upregulated in retinoblastoma [22] and malignant transformed bronchial epithelial cells [23,24], whereas it is downregulated in breast cancer [25], lung cancer[26], and cholangiocarcinoma [27]. It had also been reported that miR-320 was regulated by PTEN in mammary stromal fibroblasts [28], correlated with recurrence-free survival in colon cancer [29], inhibited proliferation in acute myelogenous leukemia [30], and suppressed prostate cancer cells by downregulating the Wnt/beta-catenin signaling pathway [31]. Lately, Iwagami et al. reported that miR-320c was upregulated in gemcitabine-resistant pancreatic cancer cells and induced the resistance to gemcitabine [32]. While further searching the putative target of miR-320c with TargetScan, we found that miR-320c targeted position 2044 on the 3′-untranslated region (UTR) of the PRDM1 gene. Therefore, the reciprocal reaction of miR-320c and PRDM1 in DLBCL remains to be elucidated. Taken together, epigenetic dysregulation is involved in the expression of PRDM1α and PRDM1β in DLBCL. Hypomethylation or demethylation of the PRDM1β promoter results in the overexpression of PRDM1β in lymphoma, while the higher or lower methylation of the PRDM1α promoter only plays a role when PRDM1α is downregulated. Therefore, a low PRDM1α/PRDM1β ratio may be a promising prognostic factor in non-GCB DLBCL. This may be helpful in understanding the mechanism of the isoform-specific roles of tumor suppressor genes in tumorigenesis. 14

Acknowledgments This work was supported by the National Natural Science Foundation of China (81071942). Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. References


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Figure Legends Figure 1. The ratio of PRDM1α/PRDM1β significantly decreased in B lymphoma cell lines and could be restored by 5-aza. (A) PRDM1α and PRDM1β expression was detected using semi-quantitative RT-PCR. Levels of genes expression are normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (B) The expression levels of PRDM1α and PRDM1β mRNA in two B-cell


lymphoma cell lines, non-tumor cell lines, and normal B cells. (C) The ratio of PRDM1α/PRDM1β in Namalwa cells was lower than in SU-DHL-4 cells. (D) The expression levels of PRDM1α and PRDM1β in SU-DHL-4 and HEK 293T cells after treatment with 5-aza. (E) The ratio of PRDM1α/PRDM1β in SU-DHL-4 cells was reversible after treatment with 5-aza. (F) The percentage of survival of Namalwa and SU-DHL-4 cells after treatment with 5-aza.

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Figure 2. PRDM1α expression in B lymphoma cell lines was related to promoter CpG island methylation. (A) One CpG island was identified at 986–1234 bp (1001 bp as the TSS) of the PRDM1α promoter region. (B) Bisulfite sequence analysis of PRDM1α was performed. Eight clones were sequenced for each cell line. White and black circles represented unmethylated and methylated alleles,


respectively. (C) The methylation frequency in each cell lines.

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Figure 3. PRDM1β expression in B lymphoma cell lines was related to promoter CpG island methylation. (A) One CpG island was identified at 826–1025 bp (1001 bp as the TSS) of the PRDM1β promoter region. (B) Bisulfite sequence analysis of PRDM1β was performed. Eight clones were sequenced for each cell line. White and black circles represented unmethylated and methylated alleles,


respectively. (C) The methylation frequency in each cell lines.

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Figure 4. Promoter activity of CpG island of PRDM1α and PRDM1β and its regulation by in vitro methylation. Plasmid constructs containing various lengths of the 5′-flanking region of the PRDM1α (A) and PRDM1β (B) genes were cloned into pGL4.15-Basic vector and used for luciferase assays. (C) The in vitro methylation was performed on the plasmid constructs of the highest transcriptional activity of PRDM1α and PRDM1β CpG islands and confirmed by restriction


endonuclease HpaII. (D) The luciferase assays were further carried out on plasmid constructs after methylation. The values for luciferase activity were normalized by the activity in cells transfected with pGL4.15-Basic vector without insert.

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Figure 5. PRDM1α and PRDM1β promoter CpG islands methylation in diffuse large B-cell lymphoma (DLBCL). Heat map showed distinct methylation profiles of PRDM1α (A) and PRDM1β (B) promoter CpG island regions in cells lines, PRDM1β-positive (β+), PRDM1β-negative (β-) tumor samples of DLBCL and reactive hyperplasia (RH). The methylation frequency of PRDM1α promoter for the 31 CpG dinucleotides (C) and PRDM1β promoter for the 11 CpG dinucleotides (D) in β+, β-,


and RH cases. ***P < 0.0001, **P < 0.01. (E) Methylation frequency of specific CpG dinucleotides, CpG 7 and CpG 16–21, compared with the remaining CpGs in the sample pool (others). Boxes represent mean ± SD. *** P < 0.0001, ** P < 0.01. The methylation frequency of specific CpG dinucleotides, CpG 7 (F) and CpG 16–21 (G), in β+, β-, and RH cases, respectively. ** P < 0.01, * P < 0.05.

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Figure 6. Methylation involved in regulating the differential expression level of microRNAs in DLBCL. (A) Hierarchical clustering of differentially expressed miRNAs in Namalwa cells absence (Con) or presence (Add) of 5-aza on the basis of all human miRNAs spotted on the chip. Red indicates high relative expression, and green indicates low relative expression. The top three upregulated (B)


and downregulated (C) miRNAs relative expression level in β+, β-, and RH cases. ** P < 0.01.

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Supplement Figure 1. The PRDM1α and PRDM1β transcripts schematic and the relative positions of their CpG islands, and RT-PCR and BSP primer position. The opposite black arrows


indicated the RT-PCR primers, and the gray arrows showed BSP primers.

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