A total of 45 patients with newly diagnosed MM [24 males, 21 females; median age, 55 years (range, 45–75 years)] were recruited from the Department of Hematology, Affiliated Union Hospital of Fujian Medical University (Fuzhou, China). The diagnosis of MM was based on standard criteria [International Myeloma Working Group, 2003 (22)], and the presence of >60% plasma cells in the bone marrow. A total of 18 healthy donors [10 males, 8 females; median age, 50 years (range, 45–68 years)] were recruited as a control group. The study was approved by the Institutional Review Board of Affiliated Union Hospital of Fujian Medical University, and informed consent was obtained from all subjects prior to sample collection.

Mononuclear cells (MNCs) were isolated from patient bone marrow samples using Ficoll-Hypaque density gradient centrifugation (Inno-Train Diagnostik GmbH, Kronberg im Taunus, Germany). Total RNA was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). To determine miR expression, total RNA (1 µl/sample) was reverse-transcribed using miR-specific stem-loop RT primers, reverse transcriptase, RT buffer, dNTPs and an RNase inhibitor, according to the manufacturer's protocol (TaqMan^® MicroRNA Reverse Transcription kit; Applied Biosystems; Thermo Fisher Scientific, Inc.). qPCR was performed using an Applied Biosystems™ StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific, Inc.). The 20-µl reaction mixture contained the corresponding complementary DNA (2 µl), miRNA-specific TaqMan primers (1 µl), TaqMan Universal PCR Master mix (10 µl; Applied Biosystems; Thermo Fisher Scientific, Inc.) and double-distilled H2O (7 µl). The PCR cycling conditions were as follows: Denaturation at 95°C for 10 min, followed by 50 cycles of denaturation at 95°C for 15 sec and annealing at 60°C for 1 min. RNU6B served as an endogenous housekeeping control for data normalization of miR levels. mRNA was reverse-transcribed in RT reactions containing 500 ng total RNA extracted from samples, 2 µl 5X PrimeScript™ Buffer (Takara Bio, Inc., Otsu, Japan), 0.5 µl 1X PrimeScript™ RT Enzyme Mix I (Takara Bio, Inc.) and 0.5 µl oligo (dT) primer. The 10-µl reactions were incubated for 42 min at 37°C, followed by 30 sec at 85°C and subsequent exposure to 4°C. The endogenous mRNA levels of Bmi-1 were detected using the SYBR^® Green PCR Master mix kit (Takara Bio, Inc.), according to the manufacturer's protocol. The PCR cycling conditions were as follows: An initial denaturation step at 95°C for 15 min, followed by 40 cycles of denaturation at 94°C for 15 sec, annealing at 55°C for 30 sec and extension at 72°C for 30 sec. β-actin served as an internal control. The following primers (Invitrogen; Thermo Fisher Scientific, Inc.) were used: Forward, 5′-AAATCAGGGGGTTGAAAAATCT-3′ and reverse, 5′-GCTAACCACCAATCTTCCTTTG-3′ for Bmi-1; and forward, 5′-TTGTTACAGGAAGTCCCTTGCC-3′ and reverse, 5′-ATGCTATCACCTCCCCTGTGTG-3′ for β-actin. The comparative quantification cycle (Cq) method was used to measure the relative changes in expression; 2-ΔΔCq represents the fold-change in expression (23).

HEK-293T cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen; Thermo Fisher Scientific, Inc.) with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.). U266 and RPMI8226 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI 1640 medium (Invitrogen; Thermo Fisher Scientific, Inc.) with 10% FBS, 50 U/ml penicillin and 50 µg/ml streptomycin.

The pri-miR-203 sequence was amplified and cloned into the pWPXL lentiviral vector (donated by Dr Didier Trono, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland). The primers were as follows: Forward, 5′-GAATTCCGTCTAAGGCGTCCGGTA-3′ and reverse, 5′-GCGGCCGCGTTCCCACAGCACAGC-3′. The pWPXL vectors were transfected into HEK-293T cells with the packaging plasmid psPAX2 and the VSV-G envelope plasmid pMD2.G (donated by Dr Didier Trono) using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Cell supernatants were collected at 48 h post-transfection and passed through a 0.22-mm filter. The virus particles were harvested 48 h following transfection. The titer of purified virus was 2.0×108 IU/ml. Cells (1×105) were infected with 1×106 recombinant lentivirus-transducing units and 6 µg/ml Polybrene (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany).

Cell proliferation was measured using the Cell Counting kit (CCK)-8 assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Cells were seeded into 96-well plates (4×103/well), and 10 µl CCK-8 solution was added to 90 µl culture medium. Cells were subsequently incubated for 2 h at 37°C and the optical density was measured at 450 nm and 650 nm. Three independent experiments were performed.

For cell cycle analysis, cells were collected and fixed in ice-cold 70% ethanol overnight. The fixed cells were washed with phosphate-buffered saline (PBS) and stained with freshly-prepared PBS containing 25 µg/ml propidium iodide (Sigma), 10 µg/ml RNaseA, 0.05 mM ethylene diamine and 0.2% Triton X-100 tetra-acetic acid for 30 min in the absence of light. For each sample, at least 20,000 cells were acquired on an EPICS Altra flow cytometer (Beckman Coulter, Inc., Brea, CA, USA) and analyzed using Multicycle AV software version 5.0 (Phoenix Flow Systems, San Diego, CA, USA).

For apoptosis detection, cells (2×10⁵) were collected and stained with an Annexin V-phycoerythrin (PE)/7-aminoactinomycin D (AAD) Apoptosis Detection kit (Sigma-Aldrich; Merck Millipore) according to the manufacturer's protocol. Cells were acquired and analyzed as above.

The miR-203 mimic and negative control were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). The sequence of the RNA duplex control was 5′-UUCUCCGAACGUGUCACGU-3′. Oligonucleotide transfection was performed with Lipofectamine 2000.

The wild-type and mutant 3′UTRs of Bmi-1 were cloned downstream of a cytomegalovirus (CMV) promoter-driven firefly luciferase cassette in a pCDNA3.0 vector (Thermo Fisher Scientific, Inc.). The primers used were as follows: Forward, 5′-AGTGGACGTTCACCGAGTT-3′ and reverse, 5′-TCCCAAAAGCGCATTTATT-3′ for wild-type; and forward, 5′-CTAGAATGAAGATGTCCCCCATCTTATACCCCTAAC-3′ and reverse, 5′-GATGGGCGGACATCTTCTTTCTAGCAGGGAGACACTG-3′ for mutant. For the luciferase assay, 10,000 U266 cells were cultured in 24-well plates and cotransfected with 20 pmol RNA (negative control or miR-203 mimic), 200 ng luciferase reporter construct and 20 ng pRL-CMV renilla luciferase reporter construct. Following a 48-h incubation, luciferase activity was measured using the Dual-Luciferase^® Reporter assay system (Promega Corporation, Madison, WI, USA).

Cells were washed with cold PBS and lysed in culture dishes using PhosphoSafe™ extraction reagent (Merck Millipore) containing 1% protease inhibitor cocktail (EDTA-free; Thermo Fisher Scientific, Inc.). Protein concentrations were then determined using Bio-Rad detergent-compatible protein assays (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Proteins (50 µg) were loaded onto 12% SDS-PAGE gels, electrophoresed and transferred onto polyvinylidene difluoride membranes (0.22 µm; Merck Millipore). Membranes were blocked with 5% nonfat milk and incubated with rabbit anti-Bmi-1 monoclonal antibody (1:1,000; catalog no. 2830S; Cell Signaling Technology, Inc., Danvers, MA, USA) and rabbit polyclonal anti-β-actin (1:1,000; catalog no. ab119716; Abcam, Cambridge, MA, USA) for 1.5 h at room temperature. Following washing, membranes were incubated for 1 h at room temperature with a horseradish peroxidase-conjugated mouse anti-rabbit secondary antibody (1:1,000; catalog no. bs-0295M; Bioss Inc., Woburn, MA, USA). Protein bands were visualized using an Enhanced Chemiluminescence reagent (Pierce Biotechnology, Inc., Rockford, IL, USA).

Statistical analyses were performed using GraphPad Prism software version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Data are presented as the mean ± standard error. The data were subjected to two-tailed Student's t-test. Linear regression analysis was used to investigate the relationship between the expression of miR-203 and Bmi-1. P<0.05 was considered to indicate a statistically significant difference.

Bmi-1 is upregulated significantly in various malignancies including MM, and may be post-transcriptionally regulated by miRNAs. Therefore, Bmi-1 may be regulated by miRNAs in MM. The online miRNA target-prediction tools, TargetScan (www.targetscan.org/vert_71/), PicTar (pictar.mdc-berlin.de/) and miRanda (www.microrna.org/microrna/home.do), were used to predict Bmi-1-targeting miRNAs. The number of potential Bmi-1-targeting miRNAs was 16, 12 and 82 for TargetScan, PicTar and miRanda, respectively. Altogether, a pool of 86 miRNAs that may potentially target Bmi-1 was selected. Of these miRNAs, miR-203 has been reported as a tumor suppressor miRNA inhibiting cellular proliferation by targeting CREB1 mRNA in MM (20). Therefore, the present study focused on miR-203. To determine whether miR-203 exerts its function by downregulating the expression of Bmi-1 through direct binding to its 3′UTR, full-length fragments of Bmi-1 mRNA 3′UTR (wild-type or mutant) were constructed and inserted immediately downstream of the luciferase reporter gene. For the luciferase assays, the miR-203 mimic was cotransfected with the luciferase 3′UTR constructs into U266 cells and RPMI8226 cells. The results revealed that miR-203 decreased the relative luciferase activity when transfected with wild-type, but not mutant, Bmi-1 3′UTR (P=0.022 and P=0.016 for U266 and RPMI8226 cells, respectively; Fig. 1A). In accordance with these results, a marked decrease was observed in endogenous Bmi-1 protein (Fig. 1B) and mRNA (P=0.018 and P=0.024 for U266 and RPMI8226 cells, respectively; Fig. 1C) expression levels in U266 cells and RPMI8226 cells following infection with miR-203-expressing lentivirus. Taken together, these results suggested that miR-203 may downregulate Bmi-1 expression post-transcriptionally by directly targeting its 3′UTR.

To determine the functional role of miR-203 in MM, a lentivirus vector harboring miR-203 was constructed and used to establish two stable MM cell lines, U266-203 and RPMI8226-203. Analysis of proliferation (Fig. 2A and B) revealed that ectopic expression of miR-203 resulted in a significant decrease in the growth of U266-203 (day 3, P=0.0215; day 5, P=0.0129) and RPMI8226-203 (day 3, P=0.0198; day 5, P=0.0095) cells. As miR-203 inhibits MM cell proliferation, the effect of miR-203 on MM cell cycle progression was investigated. The cell cycle distribution demonstrated that the proportion of cells in G1 phase was significantly increased in U266-203 (P=0.0158) and RPMI8226-203 (P=0.0142) cells compared with control vector, whereas the cell population in S phase was significantly reduced in U266-203 (P=0.0307) and RPMI8226-203 (P=0.0134) cells (Fig. 2C and D). Therefore, the growth-inhibiting effect of miR-203 on MM cells may be due to the arrest of cell cycle progression at the G1/S transition.

Annexin V-PE/7-AAD staining was performed to evaluate the effect of miR-203 overexpression on MM cell apoptosis. The percentage of apoptotic U266-control and U266-203 cells was 1.5±0.008 and 36±2.04% (P<0.001), respectively (Fig. 3A); the percentage of apoptotic RPMI8226-control and RPMI8226-203 cells was 13.66±0.98 and 30.06±1.65% (P=0.014), respectively (Fig. 3B). Therefore, ectopic expression of miR-203 may promote MM cell apoptosis by targeting Bmi-1.

To determine miR-203 expression levels in bone marrow MNCs from newly diagnosed MM patients and healthy donors, RT-qPCR was performed. miR-203 was significantly downregulated in MM patients compared with healthy controls (P<0.001; Fig. 4A). Bmi-1 was relatively highly expressed in MM and negatively correlated with miR-203 expression (P=0.0128; Fig. 4B). Furthermore, miR-203 expression levels were examined in certain patients following treatment remission and relapse. miR-203 expression increased following remission and declined again following relapse (Fig. 4C). Taken together, these results demonstrated that the expression levels of miR-203 were downregulated in MM patients, and may be associated with the current disease state.