The present study was approved by the Ethics Committee of Central South University, Changsha, China.

A total of 68 pairs of MM tissues and adjacent normal tissues were collected. The clinicopathological information is summarized in Table I. These MM patients received neither radiation therapy nor chemotherapy before surgical resection. Written informed consents had been obtained. Tissues were snap-frozen in liquid nitrogen, then stored at −80°C before use.

Human embryonic kidney HEK293 cells, MM B16 and A375 cell lines, and normal skin HACAT cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured in RPMI-1640 medium (Life Technologies, Carlsbad, CA, USA) added with 10% fetal bovine serum (FBS; Life Technologies) in a 37°C humidified atmosphere of 5% CO₂.

Total RNA was extracted using TRIzol reagent (Life Technologies) according to the manufacturer's instruction. For miR detection analysis, real-time PCR was performed using PrimeScript^® miRNA RT-PCR Kit (Takara, Dalian, China), according to the manufacturer's instructions. U6 was used as the internal reference. For qPCR detection, 1 μl cDNA solution, 10 μl PCR Master Mix, 2 μl of primers, and 7 μl H₂O were mixed to obtain a final reaction volume of 20 μl. The reaction conditions were 95°C for 10 min, and 40 cycles of 95°C for 15 sec and 60°C for 30 sec. The relative expression was analyzed by the 2^−ΔΔCt method (20).

Cells were solubilized in cold RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). The proteins were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane (Life Technologies). The PVDF was incubated with phosphate-buffered saline containing 5% milk overnight at 4°C. Then, the PVDF membrane was incubated with rabbit polyclonal anti-human HIF-1α antibody (1:100, ab2185; Abcam, Cambridge, MA, USA) and rabbit monoclonal anti-human GAPDH antibody (1:200, ab181602; Abcam) at room temperature for 3 h. After washed by PBS for 3 times, the membrane was incubated with goat anti-rabbit secondary antibody (1:10,000, ab7090; Abcam) at room temperature for 1 h. After three washes by PBS, an ECL kit (Pierce Biotechnology, Rockford, IL, USA) was used to perform chemiluminescence detection. The relative protein expression was analyzed by Image-Pro^® Plus software version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA), represented as the density ratio vs. GAPDH.

Scramble miR mimic (miR-NC), miR-18b mimic, blank pcDNA3.1 vector, pcDNA3.1-HIF-1α plasmid, and blank pLVTH vector, pLVTH-miR-18b lentiviral plasmid were purchased from Amspring Co., (Changsha, China).

Cells (1×10⁵ cells/well) were seeded in 24-well plates, and cell transfection was performed using 100 nM of diluted Lipofectamine 2000 (Life Technologies), according to the manufacturer's instruction. Cells were then cultured in a cell incubator containing 5% CO₂ at 37°C, and harvested at 48 h post-transfection for further analysis.

Cells (5,000 cells/well) were cultured in a 96-well plate, each well had 100 μl fresh serum-free medium with 0.5 g/l MTT. Following incubation at 37°C for 0, 24, 48 and 72 h, the medium was removed by aspiration and 50 μl dimethyl sulfoxide was added to each well. Following incubation at 37°C for 10 min, the A570 of each sample was measured using a plate reader.

Cells (2×10⁵) were washed twice with DPBS and resuspended in 70% ethanol. After fixed overnight at −20°C, cells were pelleted, washed twice in PBS with 3% BSA and pelleted. Cells were then resuspended and incubated for 30 min at room temperature in propidium iodide staining buffer containing 3% BSA, 40 μg/ml propidium iodide, and 0.2 mg/ml RNase in PBS. DNA content analyses were carried out using the flow cytometry (FACSCalibur; Beckman Coulter).

After cultured for 24 or 48 h, the medium supernatant was collected and diluted 1:4,000 in DPBS. The amount of glucose in the supernatant was then detected using the Glucose Uptake Colorimetric assay kit (Sigma-Aldrich) in accordance with the manufacturer's protocol. The absorbance was detected at 412 nm with ELx800 type ELISA reader (BioTek Instruments, Inc., Winooski, VT, USA).

Metabolites were quantified from medium supernatant using a Lactate Assay kit (Sigma-Aldrich) after cultured for 24 or 48 h. Assays were performed according to the manufacturer's instructions. The concentrations were normalized to protein contents determined by a BCA protein assay (Pierce Biotechnology) using bovine serum albumin (Sigma-Aldrich) as a standard protein.

Targetscan software (www.targetscan.org/) was used to analyze the putative target genes of miR-18b.

Mutations of miR-18b binding sites in the HIF-1α 3′-untranslated region (UTR) were introduced using Easy Mutagenesis System kit (Promega, Madison, WI, USA), according to the manufacturer's instruction. The wild-type (WT) or mutant type (MT) of HIF-1α 3′UTR was cloned downstream of the firefly luciferase coding region of pmirGLO™ Luciferase vector (Promega), generating the WT or MT HIF-1α 3′UTR reporter plasmid, respectively. HEK293 cells were co-transfected with WT-HIF-1α-3′UTR or MUT-HIF-1α-3′UTR reporter plasmid, and miR-18b mimic or miR-NC, respectively. After cultured at 37°C for 48 h, cells were lysed using the lysis buffer (Promega), and Dual-luciferase reporter assay system (Promega) was used to detect the luciferase activity, according to the manufacturer's instruction.

In the control group, B16 cells were stably transfected with the blank pLVTH vector. In the experiment group, B16 cells were stably transfected with the pLVTH-miR-18b lentiviral plasmid. Male BALB/C-nu/nu nude mice (n=6) were injected subcutaneously in the dorsal flank with 1×10⁷ cells of each group. The mice still alive were sacrificed on day 50 after tumor implantation. Tumor volume was calculated by using the formula V (mm³) = 0.5 × a × b2 (a maximum length to diameter, b maximum transverse diameter). Tumor weight was also recorded.

Data are expressed as mean ± SD. GraphPad Prism 5 software (Graphpad Software, Inc., La Jolla, CA, USA) was used for statistical analysis. Data were analyzed by using a Student's t-test for two-group comparison and one-way ANOVA for multiple-group comparison. The contingency data were analyzed by using the Chi-squared test. P-value <0.05 was considered statistically significant.

In the present study, we first performed qPCR to examine the miR-18b level in a total of 68 pairs of MM tissues and their matched adjacent non-tumor tissues. As demonstrated in Fig. 1A, the expression of miR-18b was significantly lower in MM tissues, when compared with that in adjacent non-tumor tissues. These MM patients were then divided into two groups, low miR-18b expression group and low miR-18b expression group, according to the mean value of the miR-18b expression as the cut-off point. The association between the miR-18b expression and the clinicopathological features of MM were then analyzed. We found no significant association between the miR-18b expression and the age, gender, or lymph node metastasis (Table I). However, low miR-18b level was significantly associated with the increased tumor thickness as well as advanced tumor stage (Table I), suggesting that the downregulation of miR-18b may play a role in MM progression. In addition, we then examined the miR-18b level in MM B16 and A375 cell lines. Human normal skin HACAT cells were used as control. As shown in Fig. 1B, the expression of miR-18b was also markedly reduced in B16 and A375 cells compared to HACAT cells. Therefore, miR-18b was significantly downregulated in MM.

We further used B16 and A375 cells to study the role of miR-18b in MM. B16 and A375 cells were transfected with miR-NC or miR-18b mimic, respectively. Real-time qPCR data showed that the miR-18b level was significantly increased after transfection with miR-18b mimic, but not with miR-NC, compared to the control group (Fig. 2A and B). We further performed MTT assay to examine cell proliferation, and found that overexpression of miR-18b significantly decreased the proliferation of B16 and A375 cells compared the control group (Fig. 2C and D).

As cell cycle progression is crucial for cell proliferation (21), we further examined the cell cycle distribution in each group. As indicated in Fig. 3A and B, ectopic expression of miR-18b induced a significant cell arrest at G1 stage, contributing to the suppressive effect of miR-18b on MM cell proliferation. In addition, energy metabolism is also important for cancer cell proliferation. Thus, we further examined the glycolysis level of MM cells with or without miR-18b upregulation. As shown in Fig. 3C–F, the glucose uptake and lactate secretion were significantly decreased in MM cells transfected with miR-18b mimic, indicating that the glycolysis was suppressed after miR-18b overexpression.

As HIF-1α is a key regulator in glycolysis (22), we further examined the mRNA and protein expression of HIF-1α in MM cells with or without miR-18b overexpression. No significant difference was observed in the mRNA level of HIF-1α in each group (Fig. 4A and B). However, miR-18b upregulation caused a remarkable reduction in the protein level of HIF-1α in B16 and A375 cells, when compared to the control group (Fig. 4C and D), suggesting that miR-18b negatively regulates the HIF-1α expression at a post-transcriptional level. Therefore, HIF-1α may be involved in the miR-18b-mediated MM cell glycolysis. To further reveal the relationship between miR-18b and HIF-1α, bioinformatics analysis was performed, and TargetScan software data showed a perfect base pairing between the 3′UTR of HIF-1α mRNA and the seed sequence of miR-18b (Fig. 4E), suggesting that HIF-1α is a target gene of miR-18b. To further confirm the targeting relationship between miR-18b and HIF-1α, the WT or MT of HIF-1α 3′UTR (Fig. 4F) was cloned into luciferase reporter vector, generating the WT or MT HIF-1α 3′UTR reporter plasmid, respectively (Fig. 4G). Luciferase reporter assay data showed that co-transfection with WT HIF-1α 3′UTR reporter plasmid and miR-18b mimic caused a significant decrease in the luciferase activity, which was rescued by transfection with MT-HIF-1α 3′UTR reporter plasmid (Fig. 4H). Accordingly, HIF-1α is a direct target gene of miR-18b, and the protein expression of HIF-1α is negatively regulated by miR-18b in MM cells.

We further studied whether HIF-1α was involved in the miR-18b-mediated MM cell proliferation and glycolysis. PcDNA3.1-HIF-1α plasmid was transfected into the miR-18b-overexpressing B16 and A375 cells. After transfection, the protein level of HIF-1α was significantly increased (Fig. 5A and B). MTT assay was then conducted to examine the cell proliferation. Our data showed that the proliferation was higher in B16 and A375 cells co-transfected with miR-18b mimic and HIF-1α plasmid, when compared to miR-18b-overexpressing B16 and A375 cells (Fig. 5C and D). The glycolysis level was then analyzed. As indicated in Fig. 5E–H, the glucose uptake and lactate secretion were also higher in MM cells co-transfected with miR-18b mimic and HIF-1α plasmid, when compared to cells transfected with only miR-18b mimic. Therefore, our data demonstrate that overexpression of HIF-1α rescued the inhibitory effect of miR-18b on MM cell proliferation and glycolysis.

To further reveal the role of miR-18b in MM in vivo, pLVTH-miR-18b lentiviral plasmid was stably transfected into B16 cells. After transfection, the miR-18b level was significantly increased compared to the control B16 cells transfected with blank pLVTH vector (Fig. 6A). Then, nude mice were subcutaneously implanted with these cells. The mice were sacrificed on day 50 after implantation and the xenografts were obtained (Fig. 6B). The survival curve data indicated that overexpression of miR-18b decreased the death rate caused by tumor growth (Fig. 6C). The tumor volume and weight were then determined. As indicated in Fig. 6D and E, the tumor volume and weight were significantly decreased in the miR-18b overexpression group compared to the control group. We also examined the protein expression of HIF-1α in the tumor xenografts. Our data showed that the protein level of HIF-1α was also lower in the miR-18b overexpression group (Fig. 6F). Therefore, our findings suggest that miR-18b has a suppressive effect on the MM growth in vivo, probably through inhibition of HIF-1α-mediated glycolysis.