Patient samples
One hundred and twenty formalin fixed, paraffin-embedded (FFPE) or fresh frozen diagnostic lymph node biopsy tissues including 93 B-cell NHL and 27 T-cell NHL were obtained from five hospitals in Hong Kong (Queen Mary Hospital, Kwong Wah Hospital, Princess Margaret Hospital, United Christian Hospital and Pamela Youde Nethersole Eastern Hospital). Among B-cell NHL cases, there were 56 DLBCL cases, 28 mantle cell lymphoma (MCL) cases, 1 FL cases, 5 Burkitt's lymphoma (BL) cases and 3 small lymphocytic lymphoma (SLL) cases. Among T-cell NHL, there were 20 peripheral T-cell lymphoma (PTCL) and 7 angioimmunoblastic T-cell lymphoma (AITL) cases. The diagnosis of NHL was made according to the WHO (World Health Organization) classification [21]. Eleven FFPE tonsils tissue were collected from healthy individuals undergoing tonsillectomy. Samples were obtained with informed consent. Our study was approved by the Institutional Review Board of Queen Mary Hospital and in accordance with the Declaration of Helsinki.
Cell culture
Five MCL cell lines (GRANTA-519, JEKO-1, MINO, REC-1, and SP53), two DLBCL cell lines (SU-DHL-6 and SU-DHL-16), two ALK (+) anaplastic large cell lymphoma (ALCL) cell lines (KARPAS-299 and SU-DHL-1) and one T-cell lymphoblastic lymphoma cell line (SUP-T1) were used in this study. REC-1 and SP53 were kind gifts obtained from Prof Raymond Lai (Department of Laboratory Medicine and Pathology, University of Alberta and Cross Cancer Institute). Other cell lines were purchased from Deutsche Sammlung von Mikroogranismen und Zellkulturen (DSMZ) (Braunschweig, Germany). Cell lines were maintained in RPMI-1640 medium (DMEM for GRANTA-519), supplemented with 10-15% fetal bovine serum, 50U/mL of penicillin and 50ug/mL streptomycin in a humidified atmosphere of 5% CO2 at 37℃. All cell culture reagents were obtained from Invitrogen (Carlsbad, CA, USA).
DNA and RNA extraction
DNA Blood Mini kit (Qiagen, Hilden, Germany) was used to extract DNA from NHL cell lines and healthy peripheral blood. Automated DNA extraction system (DNA Tissue Kit from Qiagen) was employed to extract DNA from NHL patient frozen biopsies. DNA isolation from FFPE NHL patient samples and normal tonsils was extracted with QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). Extraction of total RNA was performed with Direct-zol™ RNA MiniPrep kit (Zymo Research).
Methylation-specific polymerase chain reaction (MSP)
DNA bisulfite treatment was performed for conversion of unmethylated cytosine into uracil with EpiTect Bisulfite Kit (Qiagen, Hilden, Germany). MSP primers were designed at the CpG island embedded at the promoter region of miR-1250 host gene AATK (Figure 1a). The enzymatically methylated control DNA (Chemicon/Millipore, Billerica, MA, USA) was used as positive control for methylated-MSP (M-MSP) and negative control for unmethylated-MSP (U-MSP). Details of primer sequence and PCR condition for MSP were given in Table 1.
Table 1. Primer sequences and PCR reaction conditions for AATK/miR-1250
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Forward primer (5’ to 3’)
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Reverse primer (5’ to 3’)
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Tm/cycles/MgCl2
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Reference
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Methylation-specific PCR (MSP) for AATK/miR-1250
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M-MSP
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TCG GAT TGT ATT AGC GGA GTT TC
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CGC CGC AAA TAC GAA ACG
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57℃/35x/2 mM
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NA
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U-MSP
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TTT GGA TTG TAT TAG TGG AGT TTT
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ACC ACC ACA AAT ACA AAA CA
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55℃/37x/2 mM
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NA
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Quantitative real-time reverse transcription-PCR
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AATK
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CGG GTT CAA GGA GTT TGA GA
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GTG AGT GGC AGG ACG TAC AC
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NA
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NA
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WDR1
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CAG TGT CTG ACG GTG CAT AA
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ACG TCC AGT TTC ACA ACT CC
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NA
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[23]
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GAPDH
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ACC ACA GTC CAT GCC ATC ACT
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TCC ACC ACC CTG TTG CTG TA
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NA
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[22]
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β-Actin
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GGA CTT CGA GCA AGA GAT GG
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AGC ACT GTG TTG GCG TAC AG
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NA
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[24]
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Cloning of luciferase reporter constructs with SRBSs of miR-1250-5p
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MAPK1
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AAG GGC TAG CTT GTG TCC CTG TAT TAC CAA AA
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AGG GTC GAC TCT GGG GAA CAT AAC GAA GG
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55℃/35x/2 mM
|
NA
|
WDR1
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AAG GGC TAG CCC TTC CTT TTC TTT TTC AGT GC
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AGG GTC GAC GCG CTC AAA GTG TTT TCA CA
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55℃/40x/2 mM
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NA
|
Key: M-MSP, methylated MSP; U-MSP, unmethylated MSP; Tm, annealing temperature; SRBS, seed region binding site.
Quantitative real-time reverse transcription-PCR (qRT-PCR)
miR-1250-5p was quantified by TaqMan MicroRNA Reverse Transcription Kit and TaqMan MicroRNA Assay Kit (ABI, Foster City, CA, USA) according to the manufacture’s protocol. RNU48 was used as reference. MAPK1 (Hs01052196_m1, ABI) was quantified by Taqman Gene Expression Assay (ABI, Foster City, CA, USA) and normalized to GAPDH (Cat. Hs00266705_g1). AATK and WDR1 gene were quantified by SYBR Green Master Mix (ABI, Foster City, CA, USA). GAPDH and β-Actin were used as the endogenous control for AATK and WDR1 respectively. 2−ΔΔCt method was used to analyze the expression changes of miR1250-5p and AATK before and after 5-AzadC treatment, and the expression changes of miR-1250-5p, MAPK1 and WDR1 after transfection with precursor miR-1250-5p mimics compared with scrambled oligonucleotides control in SU-DHL-1 cells. Primer sequence for AATK, WDR1, β-Actin and GAPDH were listed in Table 1 [22-24].
DNA demethylation treatment
SU-DHL-6 and SUP-T1 cells were seeded at a density of 1×106 cells/ml in 25 cm2 flasks with 0.5-1.5 μM 5-aza-2’-deoxycytidine (5-AzadC) (Sigma–Aldrich, St Louis, MO) for 6 days. 5-AzadC was replaced at every 24 hours. Cells were harvested on day 6 for DNA and RNA extraction.
Transfection of miR-1250-5p
SU-DHL-1 cells (0.5×106 cells/ml) were transfected with either precursor miR-1250-5p mimics or scramble oligonucleotides negative control (Ambion, Austin, TX, USA) at final concentration of 150 nM with Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA). The transfected cells were cultured under serum-free condition for 48-72 hours.
MTS assay and trypan blue staining
MTS assay was employed to measure cellular proliferation with CellTiter 96® AQueous One Solution Cell Proliferation Assay kit (Promega, USA). Transfected cells (2.5×104 cells/well) were seeded into a 96-well plate in 100 μl medium. At 48 hours post-transfection, 20 μl MTS reagent was added into each well and incubated for 4 hours. Then the absorbance reading at 490 nm was recorded. Trypan blue dye exclusion assay was used to determine cellular viability at 48 hours after transfection under microscope. Both dead cells and viable cells in five random microscopic fields were counted. Dead cell (%) = (total number of dead cells per microscopic field / total number of viable and dead cells per microscopic field) × 100%. Each assay was repeated in triplicate from three independent transfections.
FITC Annexin-V-PI assay
Cell apoptosis was measured by FITC Annexin-V Apoptosis Detection Kit II (BD Bioscience, USA). Briefly, 1×105 cells were harvested, washed with cold PBS and resuspended in 100 μl of Binding Buffer. Then 5 μl of Annexin-V-FITC and 5 μl of PI were added to samples. The cells were incubated for 15 mins at room temperature in dark. Lastly, 400 μl of binding buffer were added to each sample. Samples were analyzed by flow cytometry (Beckman Coulter Cytomics FC 500). Apoptotic cells included cells in early apoptosis phase (FITC Annexin V positive, PI negative) and late apoptosis phase (FITC Annexin V positive, PI positive). Three independent experiments were performed.
Transwell migration assay
Cell migration ability was detected by transwell inserts with 8-mm-pore-size in a 24-wells format (Corning, New York, NY, USA). miR-1250-5p mimics or scramble oligonucleotides negative control labelled with Cy3 (Gene Pharma, Shanghai, China) were transfected into SH-DHL-1 cells according to the protocol previously mentioned. At 48 hours post-transfection, 1×106 transfected cells were resuspended in 100 μl of RPMI 1640 medium containing 2.5% FBS and then seeded into the upper chambers. A total of 600 μl of RPMI 1640 medium with or without SDF-1α (50ng/ml) was added to the lower chamber. The plate was incubated at 37℃ in 5% CO2. After 3 hours of incubation, the cells labelled with Cy3 in the lower chamber were counted by Flow Cytometry (NovoCyte Quanteon™) and expressed as number of Cy3-postive cells per 200 μl. The transwell assay were repeated in three independent experiments.
Western blotting
SU-DHL-1 cells transfected with either precursor miR-1250-5p mimics or scramble negative control were harvested at 72 hours post-transfection and lysed in RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) with protease inhibitors and phosphatase inhibitor Cocktail (Cell Signaling Technology). Cell lysates containing 10 μg protein were separated on Mini-PROTEAN TGX™ 10% SDS-PAGE gel (Bio-Rad, Hercules, CA, USA) and transferred to 0.45 μm PVDF membranes (GE Healthcare, Chicago, IL, USA). The membranes were blocked and then incubated with anti-ERK1/2 (1:1000, Cell Signaling Technology), anti-pERK1/2 (1:1000, Cell Signaling Technology), anti-WDR1 (1:1000, Abcam) and anti-actin (1:5000, Cell Signaling Technology) at 4°C overnight. Then membranes were washed for three times and incubated with Anti-rabbit IgG, HRP-linked Antibody (1:3000, Cell Signaling Technology) for 1 hour at room temperature with gently shaking, followed by detection of protein signals with X-ray film.
Plasmid constructs
The 3’-UTRs of MAPK1 and WDR1 include the putative SRBS of miR-1250-5p (Position: 3934-3941nts of 3’-UTR for MAPK1, 895-901nts of 3’-UTR for WDR1). A 3’-UTR DNA segment of MAPK1 or WDR1 (100~200 bp) containing SRBS of miR-1250-5p was amplified and cloned into the NheI and SalI sites of a dual firefly/renilla luciferase reporter vector, pmirGLO (Promega). MAPK1 or WDR1 3’-UTR mutants with deletion of putative SRBS of miR-1250-5p were synthesized as gBlocks Gene Fragments (Integrated DNA Technologies, Coralville, IA, USA). The sequences of PCR primers and PCR conditions were summarized in Table 1.
Luciferase assay
In 24-well plate, 0.5 μg wild-type or deletion mutant plasmids and 10 nM precursor miR-1250-5p mimics or scrambled oligonucleotides negative control were co-transfected into HeLa cells (kindly provide by Dr Zou, Department of Medicine, The University of Hong Kong) using Lipofectamine 2000 transfection reagent. At 48 hours post-transfection, the luminescent signal was quantified by Dual-Luciferase Reporter Assay System (Promega) by CLARIOstar (BMG Labtech). Firefly luciferase activity was normalized by Renilla luciferase activity. Each experiment was conducted in triplicate from three independent transfections.
Statistical Analysis
The mean expression of miR-1250-5p or AATK between methylated and unmethylated NHL cell lines were compared by Student’s t-test. The differences of trypan blue exclusion assay, MTS assay, FITC Annexin-V-PI assay and transwell migration assay between SU-DHL-1 cells transfected with miR-1250-5p mimics and scrambled oligonucleotides control were compared by Student’s t-test. The difference of AATK/miR-1250 methylation frequency in different subtypes of NHL primary samples was analyzed by χ2 test. All P-values were 2-sided. P < 0.05 was considered as significant difference.