2.1 Patients and bone marrow samples
Patients’ bone marrow samples were collected between July 2010 and June 2018, from the Department of Hematology, Qilu Hospital of Shandong University, Jinan, China. The patients were newly diagnosed CML-CP (n=42) and CML-BP (n=15). Mononuclear cells were isolated from the samples by Ficoll-Hypaque density-gradient centrifugation and stored at -80°C. The study was approved by the Ethics Committee of Qilu Hospital of Shandong University, and also accorded with the Helsinki Declaration of 1975, as revised in 1983.
2.2 Cell lines and cell culture
The human cell line K562, HL60 and HEK-293 were cultured at 37 °C, 95% air and 5% CO2 in RPMI 1640, containing 10% heat-inactivated fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and without antibiotics. The cells were cultured on 6,12 and24-well plates for 18 to 24 h before the start of the experiments.
MiR-181d mimics (miR10002821-1-5)/inhibitor (miR20002821-1-5; Ribobio, Guangzhou, China) and p65 siRNA (SASI_Hs01_00171095; Sigma-Aldrich, USA) were transfected into the cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and according to the manufacturer's protocol. Plasmids, containing the RBP2 wild-type, the RBP2-mutant (defective in demethylase activity (RBP2 H483A, Addgene), p65 (laboratory owned) and the control plasmid (laboratory owned), were transfected using the Roche Transfection Reagent (Roche, Switzerland).
2.4 RNA extraction and qRT-PCR
Total RNA was extracted from the human bone-marrow samples and the cells using different treatments of Trizol reagent (Invitrogen, Carlsbad, CA, USA). Subsequently, the extracted RNA was reverse transcribed using PrimeScript RT reagent Kit using the gDNA Eraser (Takara, Japan). The cDNAs were then subjected to SYBR Green-based real-time PCR analysis. RBP2 and p65 mRNA levels were normalized to that of the human β-actin. The mRNA level of the mature miR-181d was normalized to that of U6. The probes for RBP2 used were Hs00231908_m1 (Applied Biosystems). The primers for miR-181d and U6 were MQP-0101 and MQP-0201 (Ribobio). The other primers used in qRT-PCR assays are listed in Table 2 and the expression was calculated by the 2-ΔΔCt method.
2.5 Western blot analysis
Total proteins were extracted using a lysis buffer, containing protease inhibitors, and subjected to quantitative determination. The proteins were separated by SDS-PAGE gels, transferred to PVDF membranes (Millipore, Bedford, MA) and overnight probed, at 4 °C, with specific primary antibodies against RBP2 (1:1000, Abcam), p65 (1:5000, Abcam) and β-actin (1:10000, Sigma) and followed by 1h incubation with a horseradish peroxidase-labeled goat-anti-rabbit/mouse IgG (1:6000, Abcam). Subsequently, immunoblots were probed with ECL detection reagent (Millipore) and according to standard protocols.
The mononuclear cells, that were isolated from patient bone-marrow samples, were used to prepare cytospins with glass slides fixed by a polyformaldehyde fixation solution. The samples were stained with anti-RBP2 antibody (1:150, Abcam) and anti-p65 antibody (1:150, Abcam) overnight at 4°C, followed by an incubation with a horseradish peroxidase-conjugated secondary antibody for 30 min.
Parafﬁn embedded slides were deparafﬁnized, rehydrated and subjected to antigen-retrieval using citric acid buffer. The endogenous peroxidase was deactivated by H2O2. The slides were blocked using a 10% goat serum solution and incubated with the corresponding primary antibodies overnight at 4°C. The used antibodies were: anti-RBP2 antibody (1:150, Abcam), anti-p65 antibody (1:150, Abcam) and anti-Ki67 antibody (1:100, Abcam). Next, the slides were incubated with a secondary antibody, followed by a colorimetric detection using a DAB staining kit (Vector Laboratories, USA).
2.8 Cell proliferation assay
The 5-ethynyl-2’-deoxyuridine (EDU) assay was used to detect proliferative rates of K562 and HL60 cells. The treated cells were incubated with EDU for two hours before fluorescence detection. Then, the cells were smeared on glass slides, fixed with 4% paraformaldehyde for 30 minutes and then stained using a Cell-Light™ EDU Apollo®488 In Vitro Imaging Kit (RioBio, China), and following the manufacturer’s instructions. The slides were examined by confocal laser scanning microscopy.
2.9 Chromatin immunoprecipitation (ChIP) assay
For the ChIP assay, the Cell Signaling ChIP assay protocol was used. The precipitated DNA samples were detected by PCR. The PCR primers for p65 and miR-181d promoters are listed in Table 2.
2.10 Luciferase reporter assay
MiR-181d mimics/inhibitor (Ribobio, Guangzhou, China), RBP2 wild-type/mutant 3’UTR and the internal control vector TK plasmids were transfected into HL60 and HEK-293 cells. The plasmids containing RBP2 wild-type, RBP2-mutant, defective in demethylase activity (RBP2 H483A), p65 promoter wild or binding site mutant plasmids, and the internal control vector TK plasmid, were transfected into HL60 and HEK-293 cells. The p65 expression plasmid/siRNA, miR-181d promoter wild, or binding site mutant plasmids, and the internal control vector TK plasmid were transfected into HL60 and HEK-293 cells. After 24 or 48 h of incubation, luciferase activity was measured using a Luciferase Assay System (Promega, Madison, WI, USA) and according to the manufacturer's protocol.
2.11 Tumor xenograft model
For the xenograft model, 6 NOD/SCID male mice (Hua Fu Kang Biological Technology, Beijing, China) were treated with 2Gy dose of radiation. After 24 hours, 1× 106 K562 cells were subcutaneously injected into the right or left flank of the mice. Tumor growth was monitored every 3 days. From the seventh day, miR-181d antagomir (miR30002821-4-5) or control, were injected into the tumor every 3 days. The total period lasted 16 days. All animal procedures were approved by Qilu Hospital of Shandong University Research Ethics Committee. The animal study also accorded with the ARRIVE guidelines .
2.12 Statistical analysis
All experiments were repeated at least three times. The data were expressed as mean ± standard deviation. Student’s t-test was used to compare the means between the two groups using the GraphPad Prism for Windows, version 5.00 (GraphPad Software, La Jolla, CA, USA). The p-values of < 0.05 were considered statistically significant.