Ethics statement
The study got the approval of the Clinical Ethical Committee of The Second Hospital of Shanxi Medical University. Informed consent was signed by each eligible participant.
Tissue samples
Thirty-three bone marrow samples were collected from The Second Hospital of Shanxi Medical University from 2015 to 2018. All the samples were confirmed as MDS (n = 11), MDS-AML (n = 11) or cancer-free individuals (n = 11) by bone marrow puncture and/or biopsy. Bone marrow samples were extracted with human peripheral blood lymphocyte separation solution (TBD, Tianjin, China) by density gradient method. Total RNA was isolated from monocytes using RNAiso Plus reagent (Takara, Dalian, China) and reverse transcribed into cDNA using a reverse transcription quantitative polymerase chain reaction kit (Takara), and then stored at -80℃.
Experimental animals
NHD13 mice were purchased from Jackson Laboratory and C57BL/6 mice were purchased form Kunming Institute of Zoology, Chinese Academy of Sciences [SYXK (Yunnan) K2015-0003]. Mice were raised in a specific pathogen-free animal facility. Food and water were provided ad libitum. The expressions of EZH2 and EHMT2 in peripheral blood of mice were detected at 4 months old. Then peripheral blood was collected regularly and the blood condition was detected. Blood samples were collected from mice at the age of 14 months (420 days), and then all the mice were euthanized by an intraperitoneal injection of pentobarbital (800 mg/kg) [18, 19].
Cell culture
Human MDS/AML cells (SKM-1 cells) were purchased from American Type Culture Collection (Manassas, VG, USA) and cultured in Roswell Park Memorial Institute-1640 medium containing 10% fetal bovine serum in a 95% humidified air with 5% CO2 at 37℃.
The small interfering RNA (siRNA)-NC-1, si-EZH1, si-NC-2, si-EZH2, si-NC-T2 and si-EHMT2 were designed and synthesized. Overexpression vectors of EZH2 (pcDNA3.1-EZH2) and EHMT2 (pcDNA3.1-EHMT2) were constructed, with the empty vectors as loading control. Then, the constructed vectors and siRNAs were transfected into cells using Lipofectamine 2000 (Invitrogen Inc., Carlsbad, CA, USA). And SKM-1 cells were treated with PF-06726304 (15 nM; MedChemExpress, NJ, USA) or BRD4770 (5 nM; MedChemExpress) respectively, with the treated cell named as PF group and BRD group, respectively.
Cell counting kit-8 (CCK-8) assay
The treated cells were seeded into the 96-well plates (2 × 103 cells/well), and CCK-8 solution was added at 24, 48 and 72 h. The optical density of each well was measured at 450 nm. The experiment was repeated three times in each group.
Flow cytometry
SKM-1 cells or KG-1 cells under different treatments were fixed with 70% ethanol, stained with 300 mL propidium iodide (MultiSciences Biotech Co., Ltd, Hangzhou, Zhejiang, China) in the dark and detected on the flow cytometer (MoFloAstrios EQ, Beckman Coulter, Inc., CA, USA) to analyze the cell cycle.
5-ethynyl-2’-deoxyuridine (EdU) labeling assay
SKM-1 cells or KG-1 cells under different treatments were removed from the culture medium, washed with phosphate-buffered saline (PBS), incubated with EdU solution for 2 h, and then photographed under a fluorescence microscope (Olympus, Tokyo, Japan).
Colony formation assay
SKM-1 cells or KG-1 cells under different treatments were seeded into the 12-well plates (1 × 104 cells/well), and incubated at 37℃ for one week until cell colonies were observed. The colonies were stained with crystal violet and counted.
Chromatin immunoprecipitation (ChIP)
SKM-1 cells were subjected to ChIP assay referring to a previous literature [20]. Cells were detached with trypsin and counted using Millipore Scepter 2.0 Cell (Thermo Fisher Scientific, Jiangsu, China). And 1 × 106 cells were used for each treatment. Cells were incubated for 8 min in the medium, fixed with formaldehyde and then crosslinked with 1.25 M glycine for 5 min at room temperature. All chromatin preparation and ChIP reaction were carried out at 4℃. The crosslinked cells were washed with PBS-inhibitor (NaBu 20 mM), and the cell membrane was cleaved with the HighCell ChIP kit. Chromatin was prepared in TPX tube with shear buffer S1 and 1 × protease inhibitor, and then treated into fragments of about 500 bp by ultrasound. The size of the fragments was examined on agarose gel, and the cut chromatin was frozen at -80℃. ChIP reaction was performed using the Diagenode kit on the SX-8X IP STAR compact automation system (Diagenode) for all IP procedures. According to the HighCell ChIP kit protocol, IP DNA was purified using DNA Isolation buffer with 2 µg antibody (anti-H3K27me3 or anti-H3K9me1 or anti-H3K9me2) and non-immune immunoglobulin G (IgG). Each auto-ChIP sample was performed using the Auto Histone ChIP-seq kit and contained 1 µg input chromatin. The reaction lasted for 2 h. The antibody was coated with protein A-coated magnetic beads, and then incubated for 10 h at 4 °C for IP reaction. Afterwards, 25 µL system of DNA IP or DNA input (total DNA), 1 × SYBR Green Supermax (Applied Biosystems, Inc., Carlsbad, CA, USA) and TSH2B (pp-1041–500, Diagenode; positive control of methylation) promoter were used for reverse transcription quantitative polymerase chain reaction (RT-qPCR), with the sequence of DLX5 promoter region as primer.
RT-qPCR
Total RNA was extracted from cells using TRIzol one-step reagent (Invitrogen), and then the concentration and purity of RNA were determined using UV analysis and formaldehyde deformation electrophoresis. The fluorescent qPCR reaction was performed on the instructions of the RT-qPCR kit (ThermoFisher scientific). Primers (Table 1) were designed and synthesized by Sangon Biotech (Shanghai, China). Amplification curve and dissolution curve were confirmed after reaction. The relative expression of genes was calculated by 2-ΔΔCt method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal reference.
Table 1
Primer sequence for RT-qPCR
Gene | Primer sequence |
GAPDH | F: 5′-GGGAGCCAAAAGGGTCAT-3′ |
R: 5′-GAGTCCTTCCACGATACCAA-3′ |
EZH2 | F: 5′-ATGGGCCAGACTGGGAAGAAA-3′ |
R: 5′-GGAGGTAGCAGATGTCAAGGG-3′ |
EZH1 | F: 5′-ATGGAGGATTACAGCAAGATGG-3′ |
R: 5′-GGGGCCTGGGAGGGCTAAAGGA-3′ |
EHMT2 | F: 5′-ATGCGGGGTCTACCGAGAGGG-3′ |
R: 5′-AGAGAGGGTGTGGTCCGTTCTC-3′ |
DLX5 | F: 5′-ATGACAGGAGTGTTTGACAGAAG-3′ |
R: 5′-CTAATAGAGTGTCCCGGAGGCCA-3′ |
Hoxa9 | F: 5′-ATGGCCACCACTGGGGCCCTGGGC-3′ |
R: 5′-GCCCAAATGGCATCACTCGTCTTT-3′ |
Western blot analysis
Cells in each group were lysed in radio-immunoprecipitation assay buffer containing protease inhibitors (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). The lysate was centrifuged at 16000 g and 4 °C for 20 min to collect the supernatants. The concentration of protein extracted from cells was tested using the Pierce bicinchoninic acid assay kit (Beyotime, Shanghai, China). Then, the protein was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% skim milk for 2 h and cultured with the primary antibodies at 4 °C overnight. Thereafter, the membranes were cultured with the secondary antibody for 1 h, and developed and visualized using the enhanced chemiluminescence reagent. The gray value of the target band was analyzed by Image J software (National Institutes of Health, Maryland, USA). The antibodies used were as follows: H3K27me3 (1:1000, ab6002, Abcam, Cambridge, MA, USA), β-actin (1:1000, ab8227, Abcam), H3K9me1 (1:1000, ab9045, Abcam), H3K9me2 (1:1000, ab1220, Abcam), EZH1 (1:1000, ab189833, Abcam), EHMT2 (1:1000, ab185050, Abcam) and Hoxa9 (1:1000, ab140631, Abcam).
Statistical analysis
SPSS 21.0 (IBM Corp., Armonk, NY, USA) was utilized for data analysis. Kolmogorov-Smirnov test showed that the data were in normal distribution and expressed as mean ± standard deviation. The t test was adopted for analysis of comparisons between two groups. The one-way or two-way analysis of variance (ANOVA) was applied for comparisons among multi-groups. Tukey’s multiple comparison test was applied for the post hoc test after ANOVA. The p value was obtained from a two-tailed test, and p < 0.05 meant a statistical difference.