Non-Hodgkin lymphoma (NHL) consists of various lymphoid malignancies with a diverse clinical pathology and biological characteristics. Methylation of cytosine residues by DNA methyltransferases at CpG dinucleotides in the promoter region of the genes is a major epigenetic modification in mammalian genomes that can have profound effects on gene expression. The
Non-Hodgkin lymphoma (NHL) is a common hematological cancer with multiple subtypes, derived from various differentiation stages of the B cell lineage. Burkitt lymphoma (BL) is the most common NHL subtype (69%), followed by lymphoblastic lymphoma, diffuse large B cell lymphoma (DLBCL) and anaplastic large-cell lymphoma, accounting for 18.3, 10.6 and 2.1% of the cases, respectively (
Cancer cells develop acquiring a set of functional capabilities for malignant growth, such as self-sufficiency in growth signals, insensitivity to growth-inhibitory signals and evasion from apoptosis (
The aim of the present study was to analyze
The study included 7 cell lines, Hut78 (cutaneous T cell lymphoma cell line), Maver, Z138 (mantle cell lymphoma cell lines), CA46, Raji (Burkitt lymphoma cell lines), Jurkat (acute T cell lymphoma cell line) and DB (DLBCL cell line). All the cell lines, except CA46, were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA) and 1% antibiotics (Gibco-Invitrogen, Carlsbad, CA, USA). CA46 was maintained with RPMI-1640 supplemented with 20% FBS (HyClone) and 1% antibiotics (Gibco-Invitrogen). Cells were incubated at 37°C in a humid atmosphere at 5% CO2 and split every 2–3 days depending on cell density.
Raji and Jurkat cells in the logarithmic growth phase were inoculated in a 96-well plate, with 100
5-Aza dissolved in normal saline was used to verify the effect on
Genomic DNA was extracted by the E.Z.N.A® Tissue DNA kit (Omega Bio-Tek, Lilburn, GA, USA). DNA (200 ng) in a volume of 1–5
Modified DNA was subjected to two separate PCRs. MSP primers were designed to amplify the methylated or unmethylated alleles, and the Methylamp Universal Methylated DNA kit (Epigentek) was used as a positive control. Promoter meth-ylation status was analyzed by MSP using methylated and unmethylated gene-specific primers for
RNA was isolated using TRIzol (Gibco-Invitrogen), according to the manufacturer's instructions. Total cellular RNA (1
Protein was extracted from Hut78, Maver, Z138, CA46, Raji, Jurkat and DB cell lines. Protein concentrations of cells were determined using a bicinchoninic acid protein assay kit (Applygen Technologies Inc., Beijing, China). Western blot analyses were performed using the following primary antibodies: Anti-PTPL1 (1:200; sc-15356) and anti-β-actin (1:1,000; sc-130656) (both from Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Lysates (60
The formalin-fixed paraffin-embedded (FFPE) tissues of 47 DLBCL patients and 16 reactive lymph nodes (as control) were evaluated. The archived FFPE tissues were obtained from the Department of Pathology, Peking University Third Hospital (Beijing, China). The patients were diagnosed according to the criteria of the 2008 World Health Organization classification and were clinically staged according to the Ann Arbor classification. Clinical outcomes were evaluated according to the standard international criteria.
Genomic DNA of 47 patient samples and 16 reactive lymph nodes were extracted using the E.Z.N.A® FFPE DNA kit (Omega Bio-Tek). DNA (200 ng) in a volume of 1–5
Statistical analyses were carried out with Social Sciences software (SPSS, version 20.0; IBM Corp., Armonk, NY, USA). Pairwise correlations between the methylation status of DLBCL and control patients, and the germinal center phenotype (GCB) and non-GCB patients were investigated by χ 2 test or Fisher's exact test where appropriate. Statistical significance was set at the two-sided 5% comparison wise. P<0.05 was considered to indicate a statistically significant difference.
The
To evaluate the correlation between methylation of the
Further examination was performed on the PTPL1 protein. The expression of the PTPL1 protein was ubiquitously expressed at different levels in Hut78, Maver and Z138 cells, but silenced in CA46, Raji, Jurkat and DB cells (
Cell proliferation was detected using the CCK8 kit after 12, 24, 48 and 72 h treatment (
Forty-seven samples were screened and 23 samples were followed up. Among the 23 follow-up patients, there were 11 males and 12 females, with a median age of 63 years (range, 26–81 years). Of the 23 patients, 5 (21.7%) were stage I, 6 (26.1%) were stage II, 2 (8.7%) were stage III, and 10 (43.5%) were stage IV. Using the Hans classification model, 9 cases were GCB and 14 were non-GCB, with a GCB:non-GCB ratio of 1:1.5 (
Among the 47 DLBCL cases, the promoter of gene
In 9 GCB patients, the promoter of the
The Fisher exact probability method was used to evaluate the difference of the number of methylated
The aim of the present study was to identify novel methylated biomarkers in lymphoma and to explore potential new therapeutic targets. The methylation pattern of the
In addition, the present study has detected
The
By contrast, the relative increase of
In the present study, the number of DLBCL cases was less, and that of subjects lost to follow-up was greater. More cases and future molecular studies are required to determine the role of
In conclusion, the study showed that
The authors would like to thank Professor Junmin Li (Shanghai Ruijin Hospital, China) for the provision of the DB cell lines.
Representative analyses of the methylation of
Expression of
Western blott analysis of the PTPL1 protein in Hut78, Maver, Z138, CA46, Raji, Jurkat and DB cell lines, with β-actin as a control. The PTPL1 protein was ubiquitously expressed at different levels in Hut78, Maver and Z138 cells, but silenced in CA46, Raji, Jurkat and DB cells.
5-Azacytidine (5-Aza) induces growth inhibition of (A) Raji and (B) Jurkat cells lines.
Restoration of
Methylation pattern of
Clinical characteristics of 23 patients with DLBCL.
Clinical characteristic | Patients, n (%) |
---|---|
Gender | |
Male | 11 (47.8) |
Female | 12 (52.2) |
Age, years | |
<65 | 15 (65.2) |
≥65 | 8 (34.8) |
Stage | |
I | 5 (21.7) |
II | 6 (26.1) |
III | 2 (8.7) |
IV | 10 (43.5) |
Type | |
GCB | 9 (39.1) |
Non-GCB | 14 (61.9) |
DLBCL, diffuse large B cell lymphoma; GCB, germinal center phenotype.
Patients | Methylated, n (%) | Unmethylated, n (%) |
---|---|---|
DLBCL, n=47 | 28 (59.6) | 19 (40.4) |
Reactive lymphnodes, n=16 | 1 (6.3) | 15 (93.7) |
Fisher's exact test P<0.001. DLBCL, diffuse large B cell lymphoma.
Patients | Methylated, n (%) | Unmethylated, n (%) |
---|---|---|
GCB, n=9 | 2 (22.2) | 7 (77.8) |
Non-GCB, n=14 | 9 (64.3) | 5 (35.7) |
Fisher's exact test P=0.089. DLBCL, diffuse large B cell lymphoma; GCB, germinal center phenotype.