Peripheral blood and bone marrow samples were collected from 118 MM patients with a mean age of 54.18 years (51 males and 67 females) who were hospitalized at the Department of Hematology, Daqing Oilfield General Hospital from August 2009 to April 2015. Seventy-eight healthy volunteers with a mean age of 56.05 years (36 males and 42 females) were collected as normal controls. All patients were free from other coexisting malignant diseases. Patients with extramedullary myeloma were not enrolled in the present study. No local or systemic treatment had been conducted before operation. For the use of clinical samples for research, the study was approved by the Ethics Committee of Daqing Oilfield General Hospital and performed in accordance with the Declaration of Helsinki (2000). Written informed consent was obtained from all participants. Samples were freshly frozen in liquid nitrogen, and then were stored at −80°C until RNA extraction.

MM cell lines (MM.1S, MM.1R, JJN-3 and U266) and normal plasma cells (nPCs) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). All the cells were routinely cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc.) supplemented with 10% (vol/vol) FBS (Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin in a 5% CO₂ atmosphere at 37°C. Primary CD138⁺ cells were isolated and purified from bone marrow of MM patients and healthy volunteers by using CD138-coated magnetic beads (Miltenyi Biotec, Paris, France). Primary CD138⁺ cells were freshly frozen in liquid nitrogen, and then were stored at −80°C for further use.

Total RNA was extracted from cells or samples using TRIzol reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. A TaqMan™ MicroRNA Reverse Transcription kit (cat. no. 4366597; Thermo Fisher Scientific, Inc.) was used to reversely transcribe RNA into cDNA. qPCR was performed with a Mir-X™ miRNA qRT-PCR TB Green™ kit (cat. no. 638314; Takara, Beijing, China) on the Applied Biosystems 7500 Sequence Detection system (Thermo Fisher Scientific, Inc.) in triplicate with the following cycling conditions: 95°C for 10 min (initial denaturation), and then 40 cycles of 95°C for 10 sec, 60°C for 30 sec. The specific primers for HOTAIR, GAPDH, JAK2 and STAT3 were designed and synthesized by GenePharma Co., Ltd. (Shanghai, China). GAPDH was used as an internal control. The primer sequences were as follows: HOTAIR forward, 5′-CCCTCAGGTCCCTAATA-3′ and reverse, 5′-CCGCCGTCTGTAACTCT-3′; GAPDH forward, 5′-CCACTCCTCCACCTTTG-3′ and reverse, 5′-ACCACCCTGTTGCTGT-3′; JAK2 forward, 5′-ACCTGGTGAAAGTCCC-3′ and reverse, 5′-CCAATCATACGCATAAA-3′; STAT3 forward, 5′-AGACCCACTCCTTGCC-3′ and reverse, 5′-GCTCAGTCCTCGCTTG-3′. Results were normalized to the expression of GAPDH, and the 2^−ΔΔCq method (15) was used for analysis of the quantitative changes in expression of each gene.

Lentivirus for short hairpin RNA (shRNA) directed against HOTAIR (Lv-HOTAIR) and control lentiviral vector (Lv-control) were chemically synthesized from GenePharma Co., Ltd. (Shanghai, China). The sequences of the shRNAs were as follows: 5′-GAACGGGAGTACAGAGAGA-3′ (Lv-HOTAIR) and 5′-GACTGATTCGATACGCTATGT-3′ (Lv-control). Cells were plated in 6-well plates and transfected with Lv-HOTAIR or Lv-control using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. Twenty-four h post-transfection, the transfected cells were selected with 800 mg/ml G418 for at least one month. After selection and isolation of stably transfected clones, the clones were analyzed for HOTAIR expression using qPCR assay.

For analysis of cell apoptosis, the stably transfected cells were co-cultured with 50 µM DEX (cat. no. D4902; Sigma-Aldrich; Merck KGaA) for 48 h. Then, the cells were harvested and resuspended in fixation fluid. Propidium iodide (PI) (2 µl) and Annexin V-FITC (5 µl) were added to the cell suspension for 30 min at 37°C. The cell suspension was analyzed using FACSCalibur flow cytometry (BD Biosciences, USA). The percentage of early apoptotic cells was counted and compared for the cells receiving different treatments. For analysis of cell cycle distribution, at 48 h post-transfection, the cells were harvested via trypsinization, washed with cold PBS and fixed in 70% ice cold ethanol overnight, followed by staining with 50 mg/ml PI for 30 min. Finally, the samples were detected with FACSCalibur flow cytometry.

Cell viability was measured using a Cell Counting Kit-8 (CCK-8 kit) (Dojindo, USA), according to the manufacturer's instructions. Cells were seeded in a 96-well plate at 3×10³ cells/well and incubated in a 5% CO₂ and 37°C humidified incubator with 100 µl final volume. After incubation with 50 µM DEX for 24, 48, 72 and 96 h, 10 µl CCK-8 (5 mg/ml) was added to each well, and the cells were incubated at 37°C for 4 h. The absorbance at 450 nm in each well was detected using a micro-plate analyzer (Bio-Tek Instruments, USA).

Cells were lysed with RIPA buffer (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions, and the concentration of proteins was quantified using a BCA Protein Assay Kit (Beyotime, China). Equal amounts (30 µg) of proteins were separated by 10% SDS-PAGE and electrophoretically transferred onto PVDF membranes (EMD Millipore). After being blocked with 5% non-fat milk at 37°C for 2 h, the membranes were incubated with specific primary antibodies against JAK2 (cat. no. ab108596; 1:1,000), phospho-JAK2 (p-JAK2; cat. no. ab32101; 1:500), STAT3 (cat. no. ab68153; 1:500) and phospho-STAT3 (p-STAT3; cat. no. ab32143; 1:1,000) overnight at 4°C. GAPDH was used as an internal loading control. All primary antibodies were purchased from Abcam. Finally, the membranes were washed three times with TBST buffer and incubated with HRP-conjugated goat anti-rabbit immunoglobulin G (cat. no. AS014; ABclonal Technology) for 2 h at 37°C. The bands were detected using an ECL detection system (Pierce Biotechnology) and analyzed using the Image-Pro plus software (version 6.0; Media Cybernetics Inc.).

All results from three independent experiments are expressed as mean ± SD and were analyzed using Statistical Product and Service Solutions (SPSS) 17.0 (SPSS, Inc., Chicago, IL, USA). Differences among the groups were estimated by Student's t-test or one-way ANOVA followed by Student-Newman-Keuls test. Receiver operating characteristic (ROC) curves were plotted, and the area under the ROC curve (AUC) was calculated to assess the specificity and sensitivity of predicting MM patients and normal controls. P<0.05 was considered to indicate a statistically significant difference.

To explore whether HOTAIR is detectable and altered in MM, the expression of HOTAIR was examined by qPCR in our collection of peripheral blood from 118 MM patients and 78 healthy volunteers. As presented in Fig. 1A, elevated expression of HOTAIR was observed in the serum of MM patients compared with that noted in the normal controls (P<0.05). We then determined HOTAIR expression in bone marrow samples, and the results revealed that HOTAIR expression in MM patients was significantly higher than that in normal controls (P<0.05; Fig. 1B). To further test the hypothesis that serum HOTAIR was primarily released from tumor cells, the expression level of HOTAIR in primary CD138⁺ cells was also detected. The results showed that HOTAIR expression was significantly upregulated in the primary CD138⁺ cells from MM patients compared to those from the normal controls (P<0.05; Fig. 1C). Finally, HOTAIR expression was investigated in MM cell lines including MM.1S, MM.1R, JJN-3 and U266, and normal plasma cells (nPCs). The results revealed that the HOTAIR level was considerably increased in the four MM cell lines compared to this level in the nPCs cells (P<0.05; Fig. 1D). According to the relatively high levels of HOTAIR in MM.1S, U266 and MM.1R cells, and the fact that these cell lines are suitable models for MM as described previously (16,17), three cell lines were selected for further study in vitro.

To investigate the characteristic of HOTAIR as a potential diagnostic biomarker for MM patients, ROC curve was performed on data from clinical samples. As presented in Fig. 2, representation of the data showed the AUC of HOTAIR was 0.798 (95% CI, 0.732–0.865), and the sensitivity and specificity were 70.12 and 79.94%, respectively.

To further investigate the biological roles of HOTAIR in MM cells in vitro, stable inhibition of HOTAIR expression was established in MM.1S and U266 cell lines using Lv-HOTAIR. As confirmed by qPCR analysis, HOTAIR expression in MM.1S and U266 cells was significantly downregulated by Lv-HOTAIR compared with the expression in the Lv-control-treated and untransfected cells (P<0.01; Fig. 3A). These data indicated that cell transfection was successful, and Lv-HOTAIR is a suitable target to knock down HOTAIR in MM.1S and U266 cells. Then, a CCK-8 assay was performed to detect the role of HOTAIR in the cell viability of MM cells. The results showed that knockdown of HOTAIR inhibited cell viability of the MM.1S and U266 cell lines compared to the cell viability of the Lv-control groups (P<0.05; Fig. 3B and C). To determine whether the function of HOTAIR in regards to cell viability was caused by alteration of the cell cycle, flow cytometry was performed. Cell cycle analysis showed that knockdown of HOTAIR induced MM cell cycle arrest at the G0/G1 phase (P<0.05; Fig. 3D).

To investigate whether HOTAIR plays a role in the DEX resistance of MM cells, CCK-8 assay and flow cytometric analysis were carried out to investigate the influence of HOTAIR on the viability and apoptosis of MM cell lines to 50 µM DEX. As shown in Fig. 4A and B, knockdown of HOTAIR significantly decreased the chemoresistance of MM.1R cells to 50 µM DEX by reducing cell viability at 24, 48, 72 and 96 h, and inducing cell apoptosis at 48 h (P<0.05). To confirm whether the effect of HOTAIR on DEX chemoresistance was mediated by the JAK2/STAT3 pathway, qPCR and western blot analysis were performed. The results showed that the mRNA levels of JAK2 and STAT3 in the MM.1R cells demonstrated no significant difference between the Lv-HOTAIR and Lv-control groups (Fig. 5A). Compared with the Lv-control groups, as determined by western blot analysis, expression levels of p-JAK2 and p-STAT3 levels were significantly downregulated in MM.1R cells by HOTAIR inhibition; in contrast, no obvious total protein expression changes in JAK2 and STAT3 were observed between cells receiving different treatments (P<0.05; Fig. 5B). Taken together, these data suggest that JAK2/STAT3 signaling mediated the effect of HOTAIR on MM chemoresistance to DEX.