Thirty newly diagnosed MM patients, 17 remission patients and 19 normal controls were enrolled in this study. All the patients were inpatients in the Department of Hematology, Tianjin Medical University General Hospital from June 2016 to August 2017. All MM patients were diagnosed according to the Guidelines for the Diagnosis and Management of Multiple Myeloma in China (2017 revision) (15). The clinical features of all MM patients are shown in Table 1. The remission patients who received bortezomib-basic regimens for at least four cycles were defined as very good partial response, complete response or stringent complete responses. Meanwhile, supportive therapies were given in these patients, including blood transfusion and anti-infective agents. Ten milliliters of bone marrow (BM) was taken from the patients and normal controls. This study was approved by the Ethical Committee of the Tianjin Medical University. Written informed consent was obtained from the patients for publication.
Human MM cell lines RPMI-8226, OPM-2 and U266 were obtained from the Cell Culture Center, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences. All cell lines were cultured in 90% RPMI-1640 supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO2 and 95% humidity.
Magnetic-activated cell sorting (MACS)
BM mononuclear cells (BMMCs) were isolated from heparin-anticoagulated BM of MM and normal controls using Ficoll-Hypaque density gradient centrifugation. CD138+ cells were purified using the anti-CD138 mAb-conjugated MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Ten million cells were resuspended in 80 ml buffer. Then, 20 ml CD138 MicroBeads (Miltenyi Biotec) were added and incubated at 4°C in the dark for 15 min. After washing with 2 ml buffer, the cells were centrifuged at 300 g for 5 min. The cells were resuspended up in 500 ml buffer. The MS column was placed in the magnetic field of a suitable MACS separator (Miltenyi Biotec). After preparing the column by rinsing with 3´ 500 ml buffer, the cells were applied to the column. The column was washed with 1 ml buffer and all flow-through containing unlabeled cells was collected. Magnetically labeled cells were immediately flushed out by firmly pushing the plunger into the column and collected. The purity of enriched CD138+ cells isolated was evaluated by flow cytometry and was generally >90%.
RNA expression analysis
Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, California, USA). First-strand cDNA was generated using the FastQuant RT Kit (Tiangen, Beijing, China). Quantitative real-time polymerase chain reaction (PCR) was performed in triplicate with SYBR Green (Tiangen) using IQ5 PCR instrumentation (Bio-Rad, Hercules, CA, USA). Quantitative real-time PCR primers were as follows: 5′-GGCTCAACACGGACCTAGAG-3′(F), 5′-GTCAGCTCCAAAGTGTGCAA-3′(R). β-Actin was used as an internal control.
DNA isolation and methylation analysis by bisulfite sequencing
DNA was extracted from MM cell lines and normal controls with a TIANamp Genomic DNA kit (Tiangen), and concentrations of DNA were determined by a micro-ultraviolet spectrophotometer (Bio-Rad). Bisulfite sequencing was performed by Genechem (Shanghai, China). The experiment was repeated three times and the values were averaged.
RASSF10 lentivirus was purchased from Genechem, and lentivirus was transfected into MM cell lines and cells. The MOI=100, the cells were not selected using antibiotics, and Con238 (Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycin) was used as a control. The efficiency of transfection was measured by flow cytometry and inverted microscopy.
MM cell lines and cells at 72 h after transfection were analyzed for proliferation and apoptosis. Cell proliferation was determined using a Cell Counting Kit-8 (CCK-8) (Engreen, Beijing, China). Absorbance at 450 nm was read at 1, 2, 3 and 4 days using a 96-well microplate reader (BioTek, Winooski, VT, USA). The experiment was repeated three times and the values were averaged.
The proportion of cells undergoing apoptosis was measured using the Apoptosis Detection kit (BD Bioscience, San Diego, CA, USA). Cells were stained with fluorescein isothiocyanate–annexin V and propidium iodide (PI) and analyzed with a flow cytometer (FACScan; BD Biosciences, Mountain View, CA, USA). All assays were conducted in triplicate. The experiment was repeated three times and the values were averaged.
Cellular proteins were extracted in radio-immunoprecipitation assay buffer (Beyotime, Shanghai, China) and protein concentrations were determined using a BCA assay kit (Beyotime). Cell extracts (30 mg) were boiled with equal amounts of loading dye for 10 min and separated by 12% polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes (Hybond-ECL; Thermo Fisher Scientific, Shanghai, China). Membranes were blocked in Phosphate buffered saline (PBS) with 0.1% Tween 20 (PBS-T) containing 5% non-fat milk for 1 h, and incubated with primary and secondary antibodies in PBS-T containing 5% non-fat milk. The following primary antibodies were used: RASSF10 (diluted 1:1000) (Abcam, Cambridge, UK), bcl-2 (diluted 1:1000), caspase3 (diluted 1:500), GAPDH (diluted 1:1000) (Cell Signaling Technology, Danvers, MA, USA). Primary antibody incubation was carried out overnight at 4℃. The membranes were washed with wash buffer (1×PBS and 0.01% Tween-20) and incubated with anti-rabbit or anti-mouse secondary antibody. The experiment was repeated three times and the values were averaged.
In vivo tumor growth in nude mice
Female BALB/c-nu nude mice aged 4–5 weeks were purchased from Beijing Hua Fukang Bioscience Company and were housed and monitored in a pathogen-free environment. OPM-2 MM cell line and transfected cells (n=107) were prepared in 100 ml serum-free RPMI-1640 medium and injected subcutaneously into the right dorsal flank of nude mice (n=3 each group). Measurement of tumor volume and tumor quality was performed after 21 days, and tumor volume (V) was calculated using the formula: V=0.5×a×b2, where a and b represent the longer and shorter tumor diameters, respectively. At the end of each study, animals were killed and tumors were collected and fixed in formalin for hematoxylin and eosin (HE) staining and immunohistochemical staining of anti-CD138 or anti-RASSF10 antibody to assess tumor growth. CD138 and RASFF10 staining was quantified by Image J software. All procedures were approved by the Animal Ethics Committee of the Tianjin Medical University General Hospital.
Student’s t-test was conducted for two-group comparisons. For many-group comparisons, one-way ANOVA (if the data were normally distributed) or Kruskal–Wallis test (if the data were not normally distributed) was used. The data are expressed as the mean ± SEM or median. Kaplan–Meier survival curves were constructed, and difference in survival rates was tested by log-rank test. Statistical analyses were performed using SPSS version 21.0. A value of p<0.05 was considered significant.