Background
DNA methylation, which adds a methyl group to the number 5 carbon of a cytosine ring of a CpG dinucleotide, is catalyzed by DNA methyltransferase [
1,
2]. Cancers are characterized by a global DNA hypomethylation and locus-specific hypermethyla-tion of tumor suppressor gene (TSG). Based on a pathway-specific approach, multiple TSGs in pathways including cell cycle regulation, Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling, wingless-type MMTV intergration site family (WNT) signaling, and death-associated protein (DAP) kinase-associated intrinsic tumor suppression, have been shown to be inactivated by gene hypermethylation in leukemia, lymphoma and multiple myeloma (MM) [
1,
3].
MicroRNAs are short sequences (22–25 nucleotides) of non-coding RNA molecules that regulate a range of biological processes by inducing RNA degradation and/or translation inhibition of targeted mRNAs [
4]. Precise microRNA expression is commonly dysregulated in human diseases, including cancers. In carcinogenesis, of these aberrantly expressed microRNAs in malignant cells, those upregulated microRNAs which lead to targeting of tumor suppressor genes are known as oncomiRs. On the other hand, those downregulated microRNAs which originally may inactivate oncogenes are known as tumor suppressive microRNAs [
5,
6]. Recently, DNA methylation has emerged as an important mechanism in the regulation of microRNA expression, in particular, hypermethylation of microRNA gene promoters may lead to inactivation of tumor suppressive microRNAs in cancers [
7].
In human,
MIR129 is transcribed from
MIR129-1 and
MIR129-2 located on chromosome 7q32 and 11p11 respectively. A CpG island is present in the proximity of
MIR129-2 but not
MIR129-1 promoter. Moreover, loss of
MIR129 expression by
MIR129-2 methylation has been reported in gastric, endometrial, and colorectal cancers [
8‐
10], leading to upregulation of oncogenes including cyclin-dependent kinase 6 (
CDK6) and sex determining region Y-box 4 (
SOX4) mRNAs, thereby illustrating the tumor suppressive effect of
MIR129[
9‐
12].
We therefore investigated the role of MIR129-2 methylation and MIR129-mediated tumor suppression in a range of hematological malignancies including acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), and multiple myeloma (MM), together with monoclonal gammopathy of undetermined significance (MGUS), the precursor stage of MM, and MM at relapse/progression.
Discussion
In this study, we demonstrated that
MIR129-2 was hypermethylated in NHL and MM cell lines but not in normal blood or mononuclear cells, illustrating a methylation pattern similar to other epigenetically silenced tumor suppressor microRNAs, such as
MIR34A,
MIR34B/C,
MIR124, and
MIR203, in hematological cancers [
13‐
17]. This is in contrast to some methylated microRNAs, such as
MIR127 and
MIR373, which show a tissue-specific methylation pattern, with methylation occurring in both tumor cells and their normal counterparts [
18]. Moreover, methylation leading to reversible gene silencing was illustrated here with re-expression of
MIR129 upon hypomethylation of
MIR129-2. Furthermore, overexpression of
MIR129 led to decreased cell proliferation with increased cell death. These results were consistent with a tumor suppressor role of
MIR129 in lymphoma cells, similar to its effects on other epithelial cancers [
9‐
11,
19]. In particular,
SOX4, a known target of
MIR129, facilitates differentiation of lymphocytes, and has been shown upregulated in various human cancers [
20]. Indeed, herein, downregulation of
SOX4 was shown associated with upregulation of
MIR129 upon either hypomethylating treatment or overexpression in GRANTA-519 and JEKO-1 lymphoma cell lines. Taken together, the findings indicate that hypermethylation of
MIR129-2 led to reversible inactivation of tumor suppressive
MIR129 in hematological cancers. Lastly, in cell lines which showed complete methylation of
MIR129-2, there is no deletion of the
MIR129-2 locus, i.e. chromosome 11p11 [
21], and hence complete methylation in these cells suggests biallelic
MIR129-2 methylation.
Secondly, we found that
MIR129-2 methylation was frequent and appeared to be associated with poor survival in CLL patients, which warrants future prospective studies with larger number of patients. In CLL, apart from
ZAP-70 gene hypermethylation being a favourable prognostic marker, there is little information on the role of DNA methylation in the pathogenesis and clinical outcome of the disease [
22‐
26]. Furthermore, understanding of the prognostic value of microRNA and microRNA methylation in CLL remains preliminary [
14‐
16,
27‐
29]. Hence, our observation of
MIR129-2 methylation adversely impacting on survival in CLL is a novel finding. In order to establish the prognostic significance of
MIR129-2 methylation in CLL, a multivariate analysis together with Rai stage, lymphocyte counts and high-risk karyotype is required. However, the small number of patients in this cohort precluded a multivariate analysis.
In NHL, in contrast to
MIR34A,
MIR124-1, and
MIR203, which were frequently methylated in NK- or B-cell lymphoma,
MIR129-2 methylation was frequent but comparable among B-, T- or NK-cell lymphomas. However, an interesting observation was that methylation of
MIR129-2, which is localized to chromosome 11p11, was associated with methylation of
MIR124-1 (localized to 8p23) and
MIR203 (localized to 14q32). As
MIR124-1 targets
CDK6 mRNA and
MIR203 targets
ABL and
CREB mRNAs, the strong association of methylation of these microRNAs suggested collaboration of silencing of multiple microRNAs for oncogenesis [
14,
16]. Moreover, in lymphoma samples, in which both DNA and RNA were available, significantly lower expression of
MIR129 was demonstrated in primary lymphoma samples with
MIR129-2 methylation than those without, further testifying the association of microRNA silencing with microRNA hypermethylation.
In MM, MIR129-2 methylation was more frequent in MM patients at diagnosis or relapse/progression than patients with MGUS, and hence might be an important event implicated in transformation of MGUS to symptomatic MM. Despite that these samples were not CD138-sorted, the mean and median of plasma cell percentage of these MGUS samples were 4.78 and 5 respectively, and hence well within the limit of detection by the M-MSP. However, MSP performed on CD138-sorted plasma cells would be ideal, and hence the current finding warrants further studies using CD138-sorted samples. On the other hand, there was no impact of MIR129-2 methylation on OS. However, this cohort of patients was heterogeneously-treated, and hence the prognostic impact of MIR129-2 methylation remains to be verified in a cohort of uniformly-treated patients.
Conclusions
In summary, MIR129 is a putative tumor suppressive microRNA, and methylated in a tumor-specific manner, leading to reversible microRNA silencing. MIR129-2 methylation was frequent in lymphoid but uncommon in myeloid neoplasms. In CLL, MIR129-2 methylation adversely impacted on survival. In NHL, MIR129-2 methylation was associated with methylation of other tumor suppressor microRNAs. In MM, MIR129-2 methylation was probably associated with progression from MGUS to symptomatic MM. Therefore, MIR129-2 methylation is important in the pathogenesis, disease progression and prognostication in lymphoid neoplasms. The implication of MIR129-2 methylation with methylation of other tumor suppressive microRNAs in lymphomas warrants further study.
Methods
Patient samples
Diagnostic bone marrow or tissue samples were obtained in 20 ALL, 20 AML, 11 CML in chronic phase, 61 CLL, 68 NHL, 40 MGUS, 95 MM at diagnosis, and 29 MM at relapse/progression. Patient demographics were listed in Table
1.
Table 1
Patient demographics
ALL (N = 20) | Gender (M/F) | 11/9 |
| Median age (range) | 35 (13–62) years |
| MIC type (C/PB/EPB/T) | 6/10/1/3 |
AML (N = 20) | Gender (M/F) | 9/11 |
| Median age (range) | 41.5 (20–72) years |
| FAB type (M1/M2/M4/M5) | 3/14/2/1 |
CLL (N = 61) | Gender (M/F) | 44/17 |
| Median age (range)* | 65 (37–91) years |
| Rai stage (<2/≥2)* | 37/20 |
| Median lymphocyte count (range)* | 18.5 (10–236) × 109/L |
| High-risk [del(17p)/trisomy 12]‡ | 1/7 |
| Low-risk [del(13)/normal karyotype/ other karyotype abnormalities]‡ | 7/16/6 |
CML (N = 11) | Gender (M/F) | 7/4 |
| Median age (range) | 41 (22–87) years |
| Chronic phase | 11 |
MM (N = 95) | Gender (M/F) | 37/58 |
| Median age (range) | 62 (29–91) years |
| Ig type (G/A/D/LC/NS) | 57/23/3/11/1 |
| ISS stage (I/II/III)† | 16/36/27 |
NHL (N = 68) | Gender (M/F) | 38/30 |
| Median age (range) | 60.5 (17–92) |
| Ann Arbor stage (I/II/III/IV)^ | 3/4/4/18 |
| Type (ALCL/ AITL/ PTCL,NOS/ NK-T/ FL/ MZL/ MCL/ DLBCL) | 2/ 4/ 9/ 8/ 21/ 7/ 2/ 15 |
Diagnosis of leukemia and lymphoma were made according to the French-American-British Classification and WHO Classification of Tumors respectively [
30‐
33].
In the CLL group, median overall survival (OS) was 81 months for the whole group, and 102 months in those with limited, and 54 months in those with advanced Rai stage (p=0.009). Median OS of CLL patient with low/standard-risk and high-risk karyotypes were 111 months and 21 months (p<0.001).
In the NHL group, of 36 patients with data available at clinical presentation, 23 had nodal and 13 had extranodal involvement. Correlation between microRNA methylation and expression was studied in 25 primary lymphoma samples (follicular lymphoma, N=12; diffuse large B-cell lymphoma, N=13), in which both DNA and RNA were available.
The diagnosis of MGUS and MM was based on standard criteria [
34]. Complete staging work-up included bone marrow examination, skeletal survey, serum and urine protein electrophoresis, and serum immunoglobulin (IgG, IgA, and IgM) levels. In this cohort, the median OS was 44 months, and projected 10-year OS was 19.9%. The median OS were 83 months, 60 months and 23 months in those with ISS I, II and III disease respectively (p<0.001). Definitions of relapse and disease progression followed the criteria of European Group for Blood and Marrow Transplantation Registry [
35]. Briefly, “relapse” from complete remission (CR) was defined as the reappearance of the same paraprotein detected by serum/urine protein electrophoresis, appearance of new bone lesion or extramedullary plasmacytoma, or unexplained hypercalcaemia. The definition of “disease progression” from plateau phase/stable disease was the same as the definition of relapse except that “a >25% increase in paraprotein level” replaced “reappearance of the same paraprotein”. The study has been approved by Institutional Review Board of Queen Mary Hospital, and written informed consent was obtained from the patient for publication of this report and any accompanying data or images.
Cell culture
MM cell lines LP-1 & RPMI-8226 were kindly provided by Dr Robert Orlowski (Department of Lymphoma/Myeloma, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA) and WL-2 by Prof. Andrew Zannettino (Myeloma Research Programme, The University of Adelaide, Australia). NCI-H929 was purchased from American Type Culture Collection (Manassas, VA, USA). Other MM (KMS-12-PE, MOLP-8, OPM-2 and U-266) and lymphoma (SU-DHL-6, SU-DHL-16, GRANTA-519, MINO & JEKO-1) cell lines were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) (Braunschweig, Germany). Cell cultures were maintained in RPMI-1640 (IMDM for LP-1), supplemented with 10% (15% for lymphoma cell lines) fetal bovine serum, 50 U/ml of penicillin and 50 ug/ml streptomycin in a humidified atmosphere of 5% CO2 at 37°C. All cell culture reagents were purchased from Invitrogen (Carlsbad, CA, USA).
DNA and RNA extractions
DNA was extracted from primary samples, 8 MM cell lines (KMS-12-PE, LP-1, MOLP-8, NCI-H929, OPM-2, RPMI-8226, U-266 and WL-2) and 5 lymphoma cell lines (SU-DHL-6, SU-DHL-16, GRANTA-519, MINO and JEKO-1), using QIAamp DNA Blood Mini (Qiagen, Hilden, Germany). Total RNA were harvested using mirVana™ miRNA Isolation Kit (Ambion Austin, TX, USA).
Methylation-specific polymerase chain reaction (MSP)
DNA samples were treated to convert unmethylated cytosine to uracil by EpiTect Bisulfite Kit (Qiagen, Hilden, Germany). Primers and conditions for methylated-MSP (M-MSP) and unmethylated-MSP (U-MSP) of
MIR129-2 were listed in Table
2. Primers and conditions of MSP for
MIR124-1 and
MIR203 were previously described [
14,
16]. Mononuclear cell DNA from 15 healthy donors [8 bone marrow, 3 peripheral blood, 3 CD19-sorted peripheral blood, and 1 CD138-sorted bone marrow (AllCells, CA, USA)] were used as negative control, and an enzymatically methylated control DNA purchased from CpGenome Universal Methylated DNA (Chemicon/Millipore, Billerica, MA, USA) was used as positive control in all the experiments. Amplified products were then visualized by 6% non-denaturing polyacrylamide gel stained by ethidium bromide.
Table 2
Primer sequences and reaction conditions
(I) Methylation-specific polymerase chain reaction (MSP) | | |
MIR129-2
| | | | |
M-MSP | GAGTTGGGGGATCGCGGAC | ATATACCGACTTCTTCGATTCGCCG | 188 | 59°C/ 35 |
U-MSP | GAGTTGGGGGATTGTGGAT | AATATACCAACTTCTTCAATTCACCA | 189 | 55°C/ 35 |
(II) Reverse transcription-polymerase chain reaction (RT-PCR) | | |
SOX4
| GCTGGAAGCTGCTCAAAGAC | ACCGACCTTGTCTCCCTTCT | 167 | 60°C/ 40 |
GAPDH
| ACCACAGTCCATGCCATCACT | TCCACCACCCTGTTGCTGTA | 452 | 60°C/ 40 |
Sensitivity of the M-MSP
To establish the sensitivity of the MIR129-2 M-MSP, 1 μg of methylated control DNA was 10-fold serially diluted in buffer, bisulfite-treated and amplified with MIR129-2 M-MSP primers.
Quantitative bisulfite pyrosequencing
Bisulfite-treated DNA was used as template. Methylation-unbiased primer set was used to amplify the promoter region, which overlapped with the amplicon of the MSP. Forward: 5’-AGA GGG ATA GGA TAG GTA GG-3’; reverse: 5’-AAC CCT AAA ACC CAA CAA ACT AAA TCT-3’; condition: 2 mM/55°C/50X. A stretch DNA with 9–12 adjacent CpG dinucleotides was pyrosequenced by sequencing primer: 5’-GGT TTG GAG AAA TGG A-3’.
Hypomethylating treatment
JEKO-1 was homozygously methylated for MIR129-2. Cells were seeded in six-well plates at a density of 1x106 cells/ml and cultured with 0.5–1uM of 5-aza-2’-deoxycytidine (5-azadC) (Sigma–Aldrich) for 3 days.
Quantitative real-time reverse transcription–PCR (RT-qPCR)
Short mature microRNA transcripts were quantified using stem-loop RT-qPCR which is a sensitive, specific and widely-used method designed for microRNA studies [
36]. For
MIR129, RT was performed using Taqman® MicroRNA RT Kit and Taqman® MicroRNA Assay Kit (ABI, Foster City, CA, USA), according to the manufacturer’s instructions. Total RNA was reverse transcribed in 1 mmol/l dNTPs, 50 U MultiScribe™ Reverse Transcriptase, 1× RT Buffer, 3·8 U RNase Inhibitor, and 1× stem-loop RT primer at following thermal cycling condition: 16°C for 30 min, 42°C for 30 min, and 85°C for 5 min. RT-qPCR of
MIR129 was performed using 1·33 μl of 1:15 diluted RT product in 1× Taqman® Universal PCR Master Mix, and 1× Taqman® Assay at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min.
SNORD48 was used as reference for data analysis with the 2
-ΔΔCt method [
37]. Conventional RT-qPCR was used for
SOX4 transcript, RT was performed using QuantiTect Reverse Transcription Kit (Qiagen), according to the manufacturer’s instructions. RT-qPCR was performed by iQ SYBR Green Supermix (Bio-Rad), using
GAPDH as endogenous control for data analysis with the 2
-ΔΔCt method [
37]. Primers for detecting
SOX4 and
GAPDH were summarized in Table
2.
MIR129 overexpression in JEKO-1 cells
Cells at log phase were transfected with 150nM of either negative control mimic or MIR129 oligo mimic (Ambion) at a density of 106 cell/mL using X-tremeGENE siRNA transfection reagent (Roche), according to the manufacturer’s instructions.
MTT assay
Cell proliferation was determined by colorimetric quantification of purple formazan formed from the reduction of yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) by proliferating cells. Briefly, cells were seeded in a 96-well microtitre plate at 5 × 105 /well in 100 μl of medium. At the assay time point, each well was added 10 μl of 5 mg/ml MTT reagent (Sigma-Aldrich), followed by 6-hour incubation, after which 100 μl of DMSO was added. The absorbance reading at 550 nm with reference to 650 nm was recorded. Relative abundances of proliferative viable cells from three independent experiments were calculated.
Trypan blue exclusion assay
Dead cells were visualized by trypan blue staining and five random microscopic fields were counted for each sample. Dead cells (%) = (total number of dead cells per microscopic field/ total number of cells per microscopic field) X 100. Percentages of dead cells from three independent experiments were calculated.
Statistical analysis
Correlation between
MIR129-2 methylation with continuous (mean age) and categorical variables (gender, histological subtypes, lineage [B, T or NK/T] and nodal/extranodal presentation) were studied in these 68 patients by Student’s
t-test and Chi-square test (or Fisher Exact test) respectively. Overall survival (OS) was measured from the date of diagnosis to the date of last follow‐up or death. Survival was plotted by the Kaplan‐Meier method, and compared by the log‐rank test. Moreover, in 25 primary B-cell NHL samples in which both DNA and RNA were available, the mean expression of
MIR129 in methylated and unmethylated lymphoma was compared by the Student’s t-test. Association between
MIR129-2 methylation and other previously studied tumor suppressive microRNA methylation, including
MIR34A,
MIR124-1,
MIR203 and
MIR196B[
14‐
16], in MM, NHL and CLL patients were studied by Χ
2 test. The mean results from triplicate experiments after
MIR129 transfection were compared by Student’s
t-test. All p-values were 2-sided.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CSC designed the study. KYW, RLHY conducted the experiments. CCS, YLK, CYL, PKH, FC, RL helped in sample collection and clinical data retrieval. CSC, KYW, RLHY, DYJ helped in data analyses. All authors were involved in the writing and final approval of the manuscript.