Dr Jiaren Xu, Department of Geriatric Medicine, Geriatric Hospital of Nanjing Medical University, Jiangsu Province Geriatric Institute, 18 Luojia Road, Nanjing, Jiangsu 210000, P.R. China
*Contributed equally
Methylation is a fundamental regulator of gene transcription. Long non-coding RNA maternally expressed 3 (MEG3) inhibits cell proliferation in various types of cancer. However, the molecular mechanisms of MEG3 methylation in the regulation of multiple myeloma (MM) are unknown. In the present study, MEG3 upregulation was negatively associated with the International Staging System (ISS) status of the bone marrow samples of 39 patients with MM. MEG3 overexpression in an MM cell line resulted in elevated p53 expression. Furthermore, the results of methylation-specific PCR revealed that the abnormal methylation status of the MEG3 promoter region was present in eight of the 39 bone marrow samples collected. Treatment of the MM cell line with the DNA methylation inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR) resulted in tumor cell proliferation inhibition, apoptosis induction and G0/G1 cell cycle arrest. Furthermore, 5-Aza-CdR decreased aberrant hypermethylation of the MEG3 promoter and increased the expression of MEG3. However, 5-Aza-CdR exerted no effect on p53 expression. To the best of our knowledge, the present study is the first to report that the demethylation reagent 5-Aza-CdR may serve as a therapeutic agent in MM by upregulating MEG3 expression. However, the mechanism of action was independent of p53 expression.
Multiple myeloma (MM) is one of the most common types of haematological cancer, which is becoming more common in the ageing population. It is also the cause of a number of fatal outcomes (
DNA methylation, a form of epigenetic control of gene transcription, refers to cytosine methylation at position 5 in the pyrimidine ring, which can result in inappropriate silencing of genes involved in diverse biological processes, including cell proliferation, apoptosis, migration and cell cycle arrest (
Long non-coding RNAs (lncRNAs) are a class of non-coding RNA with a length of >200 nucleotides, which possess little to no capacity for protein synthesis (
In MM, Benetatos
The present study was approved by the Institutional Review Board of the First Affiliated Hospital of Nanjing Medical University. All participants provided written informed consent. Bone marrow biopsy samples were collected from 39 patients with newly diagnosed MM who were admitted to the First Affiliated Hospital of Nanjing Medical University between January 2009 and May 2014. Patient information is listed in
MM cells were isolated from bone marrow samples using CD138 microbeads and MS-columns (Miltenyi Biotec; cat. no. 130-051-301) according to the manufacter's protocol. The MM cell line, ARP1 (American Type Culture Collection), was cultured in RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin in an incubator at 37˚C with 5% CO2.
Total RNA was isolated from ARP1 cells and patient derived MM cells using TRIzol® reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's protcol. Total RNA was reverse transcribed to cDNA using the Primescipt RT Reagent kit with gDNA Eraser (Takara Biotechnology Co., Ltd.), according to the manufacturer's protocol.
qPCR was subsequently performed on a StepOne Plus™ Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using a SYBR Green qRT-PCR assay according to the manufacturer's protocol (Takara Biotechnology Co., Ltd.). The following primer pairs, designed by Primer Premier 5 (Premier Biosoft International), were used for qPCR: MEG3 forward, 5'-GGAGCTGTTGAGCCTTCAGT-3' and reverse, 5'-CAAGCCCTGTGCTTTGGAAC-3'; and β-actin forward, 5'-AGCGAGCATCCCCCAAAGTT-3' and reverse, 5'-GGGCACGAAGGCTCATCATT-3'. The following thermocycling conditions were used for the qPCR: 40 cycles of denaturation at 95˚C for 5 sec, annealing at 60˚C for 30 sec, followed by a final extension at 72˚C for 5 min. MEG3 mRNA levels were quantified according to the standard curve of MEG3 and β-actin using the 2-∆∆Cq method (
Genomic (g)DNA was extracted from ARP1 cells and patient derived MM cells using a TIANamp Genomic DNA kit according to the manufacturer's protocol (Tiangen Biotech Co., Ltd.). Subsequently, DNA bisulfite conversion was performed on the gDNA using the EpiTect Plus Bisulfite kit (Qiagen GmbH), according to the manufacturer's protocol.
The methylation status of MEG3 was determined by MSP, using a Veriti96 PCR thermocycler (Applied Biosystems; Thermo Fisher Scientific, Inc.) with Taq PCR MasterMix (Tiangen Biotech Co., Ltd.). The following primers obtained from previous studies (
ARP-1 cells were cultured in DMEM medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin in an incubator at 37˚C with 5% CO2. ARP1 cells (2.0x106/well) were plated into 6-well plates and transfected with the 4 µg pcDNA3.1-MEG3 or 4 µg pcDNA3.1-empty (provided by Professor Wei De, Nanjing Medical University) using Lipofectamine® 2000 (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. For MEG3 knockdown, the following small interfering (si)RNA sequences were used for Lipofectamine® 2000 transfection: si-MEG3, 5'-GCUCAUACUUUGACUCUAUTT-3'; and si-negative control (NC), 5'-UUCUCCGAACGUGUCACGUTT-3'. Both sequences were designed and synthesized by Shanghai GenePharma Co., Ltd.
ARP1 cells were seeded at 2x104 cells/well in 96-well culture plates and incubated with DMEM (200 µl) containing 0.1, 1, 5, 10, 50 or 100 µg/ml 5-Aza-CdR (Sigma-Aldrich; Merck KGaA) for 72 h at 37˚C. Control cells were incubated with DMEM containing PBS (20 µl). The Cell Counting Kit-8 (CCK-8) assay (Selleck Chemicals) was used to analyze cell proliferation according to the manufacturer's protocol. RT-qPCR and MSP were performed to assess the expression of MEG3 mRNA and the methylation status of the MEG3 promoter, respectively.
For restoration experiments, ARP1 cells were treated with 5-Aza-CdR (50 µg/ml for 48 h at 37˚C), followed by MEG3 knockdown. MEG3 expression was detected by RT-qPCR at 48 h post-transfection. ARP1 cell proliferation following MEG3 knockdown was analyzed by the CCK-8 assay at 0, 24, 48 and 72 h post-transfection.
ARP1 cells and patient derived MM cells were lysed and total protein was extracted using RIPA buffer (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Total protein was quantified using the Bicinchoninic Acid Protein Assay kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Subsequently, 50 µg protein/lane was separated on 10% SDS-PAGE gels by electrophoresis and then transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk for ~2 h at room temperature and then incubated with anti-p53 (1:1,000; Cell Signaling Technology, Inc. cat. no. 2524) and anti-GAPDH (1:1,000; Cell Signaling Technology, Inc.; cat. no. 5174) primary antibodies overnight at 4˚C. Membranes were washed for 1 h with TBST buffer. Following the primary incubation, membranes were incubated for 1.5 h with appropriate secondary antibodies (horseradish peroxidase conjugated goat anti-rabbit IgG H&L; 1:4,000; Abcam; cat. no. ab6721) at room temperature. Protein bands were visualized using the Chemiluminescence horseradish peroxidase substrate (cat. no. P90720; EMD Millipore) and the Molecular Imager ChemiDoc XRS+ chemiluminescence system (Bio-Rad Laboratories, Inc.). Protein expression was quantified using Image Lab software version 5.0 (Bio-Rad Laboratories, Inc.) with GAPDH as the loading control.
To analyze the cell cycle, ARP1 cells (2.0x106/well) were plated in 6-well plates and treated with a series of concentrations of 5-Aza-CdR (0, 5, 10 and 50 µg/ml). After 48 h incubation at 37˚C with 5% CO2, cells were washed with PBS and fixed with 75% cold ethanol for 24 h at -20˚C. Subsequently, the cells were washed with PBS and stained using the Cell Cycle Detection kit according to the manufacturer's protocol (Nanjing KeyGen Biotech Co., Ltd.) at room temperature for 30 min to analyze the cell cycle with FACS (Becton, Dickinson and Company).
To analyze apoptosis, ARP1 cells (2.0x106/well) were plated in 6-well plates and treated with a series of concentrations of 5-Aza-CdR (0, 5, 10 and 50 µg/ml). After 48 h treatment at 37˚C with 5% CO2, the cells were washed with PBS. Subsequently, the cells were harvested and stained using the Annexin V-FITC Apoptosis Detection kit (Nanjing KeyGen Biotech Co., Ltd.), according to the manufacturer's protocol. Cells were stained with Annexin V and PI at 4˚C for 15 min in the dark, and subjected to FACS analysis (Becton, Dickinson and Company).
For restoration experiments, ARP1 cells were treated with 5-Aza-CdR (50 µg/ml for 48 h at 37˚C with 5% CO2), followed by MEG3 knockdown. At 48 h post-transfection, cells were used for cell cycle and apoptosis analyses.
The schematic diagram of CpG islands in the human MEG3 promoter was performed using MethPrimer software (Version 1.0;
In the present study, MEG3 levels in 39 newly diagnosed MM bone marrow samples were quantified via RT-qPCR. The clinical characteristics of the patients with MM are listed in
In the 39 newly diagnosed MM samples, an abnormal methylation pattern of the MEG3 DMRs was identified in eight of the bone marrow samples. Chi-squared test was used to examine the association between MEG3 methylation status and ISS stage (
To investigate the role of 5-Aza-CdR in cell proliferation, ARP1 cells were treated with different concentrations of 5-Aza-CdR. The results of the CCK-8 assay revealed that proliferation was inhibited by 5-Aza-CdR in a dose-dependent manner (mean inhibition proportion of 0, 5, 10, 50 and 100 µg/ml for 24 h: 3.0, 38.5, 42.8, 59.6 and 61.5%; 48 h: 7.3, 45.3, 53.9, 74.3 and 79.8%; and 72 h: 6.0, 45.1, 69.5, 91.6 and 93.5%;
The present study investigated whether MEG3 regulated the expression of p53. ARP1 cells were successfully transfected with pcDNA3.1-MEG3 or pcDNA3.1-empty (
ARP1 cells treated with 5-Aza-CdR followed by MEG3 knockdown were used to investigate whether the tumor suppressive role of 5-Aza-CdR in MM cells may be due to the upregulation of MEG3. MEG3 expression levels were detected in untreated, si-NC, si-MEG3, 5-Aza-CdR, 5-Aza-CdR + si-NC and 5-Aza-CdR + si-MEG3 groups, which demonstrated that 5-Aza-CdR could upregulate MEG3 expression in ARP1 cells (
In the present study, low MEG3 expressions were associated with higher ISS stages. A previous study reported that MEG3 overexpression inhibited proliferation, promoted apoptosis and blocked the cell cycle in ARP1 cells (
MEG3 is a maternally expressed imprinted gene, which encodes an lncRNA (
The tumor suppressor function of p53 has long been recognized and p53 has been reported to be mutated, which may lead to the loss of wild-type p53 activity in the majority of human malignancies (
DNA methylation, an epigenetic regulation mechanism, serves a role in silencing MEG3 gene expression in different types of cancer (
In conclusion, the present study suggested that MEG3 may serve as a tumor suppressor by upregulating p53 levels in MM. Furthermore 5-Aza-CdR inhibited MM cell proliferation by upregulating MEG3 expression. However, this was independent of p53 expression. Further investigation into how 5-Aza-CdR affects MEG3 and why p53 expression is not altered in MM is required. Additionally, further investigation into the mechanisms of MEG3 may provide novel therapeutic targets for MM.
The authors would like to thank Professor Wei De (Nanjing Medical University) for providing the pcDNA3.1-MEG3 and pcDNA3.1-empty plasmid.
The present study was supported by the National Natural Science Foundation of China (grant nos. 81800200 and 81670199) and the Jiangsu Province's Medical Elite Program (grant no. ZDRCA2016015).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
WY and QS assisted with all experiments and wrote the manuscript. CW and XS were responsible for the flow cytometry experiments and statistical analysis. JX and LC designed the current study and critically reviewed the manuscript. All authors read and approved the final manuscript.
The present study was approved by the Institutional Review Board of the First Affiliated Hospital of Nanjing Medical University. All participants provided written informed consent.
Not applicable.
The authors declare that they have no competing interests.
MEG3 methylation status and MEG3 expression in the bone marrow samples of patients with MM. (A) MEG3 methylation status. Samples 1 and 4 exhibited an abnormal methylation status. Samples 2, 3 and 5 exhibited a normal methylation status. (B) A standard curve was used to calculate the expression of MEG3 and β-actin following reverse transcription-quantitative PCR. (C) Relative expression of MEG3 in 39 bone marrow samples of patients with MM. MEG3 expression was detected in 36 samples. (D) MEG3 expression was negatively associated with International Staging System stage. (E) Schematic diagram of CpG islands (blue area) in the human MEG3 promoter, identified using MethPrimer software. The position of CpG islands in relation to the MEG3 transcription starting site and MSP primers were depicted. MEG3, maternally expressed 3; MM, multiple myeloma; PC, positive control; M, methylated band; U, unmethylated band; MF1, methylated forward 1; MR1, methylated reverse 1; UF1, unmethylated forward 1; UR1, unmethylated reverse 1.
5-Aza-CdR restores the normal MEG3 methylation status and MEG3 expression of ARP1 cells. (A) MEG3 methylation status and (B) expression of ARP1 cells. 5-Aza-CdR, 5-Aza-2'-deoxycytidine; MEG3, maternally expressed 3; NC, negative control; PC, positive control; M, methylated band; U, unmethylated band; 1, 0 µg/ml 5-Aza-CdR; 2, 5 µg/ml 5-Aza-CdR; 3, 10 µg/ml 5-Aza-CdR; 4, 50 µg/ml 5-Aza-CdR.
5-Aza-CdR functions as an anti-tumor factor in the ARP1 cells. (A) 5-Aza-CdR inhibited the proliferation of the ARP1 cell line. (B) Representative images of the flow cytometry analysis of the (B) apoptosis and (C) cell cycle of ARP1 cells following 5-Aza-CdR treatment. (D) 5-Aza-CdR promoted the apoptosis of the ARP1 cell line. (E) 5-Aza-CdR arrested the cell cycle at G0/G1 in the ARP1 cell line. 5-Aza-CdR, 5-Aza-2'-deoxycytidine.
Effect of MEG3 overexpression and 5-Aza-CdR on p53 expression in ARP1 cells. (A) MEG3 overexpression efficiency in ARP1 cells transfected with pcDNA3.1-empty and pcDNA3.1-MEG3. (B) MEG3 overexpression increased the protein levels of p53 in the ARP1 cell line; (C) 5-Aza-CdR exhibited no effect on p53 protein expression. MEG3, maternally expressed 3; 5-Aza-CdR, 5-Aza-2'-deoxycytidine.
MEG3 knockdown reverses the antitumor effects of 5-Aza-CdR. Analysis of (A) MEG3 mRNA expression, (B) cell proliferation, (C) representative plots and (D) quantification of apoptosis. (E) Representative plots and (F) quantification of the cell cycle in ARP1 cells treated with 5-Aza-CdR followed by MEG3 knockdown. MEG3, maternally expressed 3; 5-Aza-CdR, 5-Aza-2'-deoxycytidine; si, small interfering RNA; NC, negative control.
Distribution of variables of patients with multiple myeloma.
Variable | Number (%) |
---|---|
Median age (range) | 61 (36-82) |
Sex | |
Male | 26 (66.7) |
Female | 13 (33.3) |
Subtypes | |
IgG | 18 (46.2) |
IgA | 11 (28.2) |
Light chain | 10 (25.6) |
Durie-Salmon stage | |
I | 4 (10.3) |
II | 5 (12.8) |
III | 30 (76.9) |
International Staging System stage | |
I | 5 (12.8) |
II | 14 (35.9) |
III | 20 (51.3) |
Serum creatinine (µmol/l) | |
>176.8 | 22 |
≤176.8 | 12 |
Serum calcium (mmol/l) | |
>2.98 | 31 |
≤2.98 | 3 |
N/A | 5 |
N/A, not detected.
Distribution of methylation status among the International Staging System stages in patients with multiple myeloma.
International staging system stage | ||||
---|---|---|---|---|
Methylation status | I | II | III | P-value |
Unmethylated | 4 (12.9) | 12 (38.7) | 15 (48.4) | 0.748 |
Methylated | 1 (12.5) | 2 (25.0) | 5 (62.5) |