Serum samples (10 ml) were obtained from patients with MM (n=55; age range, 49–86 years old; 29 female patients and 26 male patients) and healthy donors (n=40) at the Anhui No. 2 Provincial People's Hospital (Hefei, China) between March 2017 and January 2020. The inclusion criteria were as follows: i) The patients were diagnosed with MM by pathological examinations, which were in accordance with the updated 2014 International Myeloma Working Group criteria for MM diagnosis (32); and ii) the clinical data for MM were complete. The exclusion criteria included patients who had received chemotherapy before the operation. The patients were selected according to the aforementioned conditions with an eventual sample size of 55. In addition, all MM patients were divided into a low group (n=27) and high group (n=28), according to the median expression level of lncRNA DANCR (0.491), as shown in Table I. This study was approved by the Ethics Committee of Anhui No. 2 Provincial People's Hospital (approval no. 2020-16-27), and adhered to the guidelines of the Declaration of Helsinki. Written informed consent was obtained from all participants.

Total RNA was extracted from serum samples and cells using TRIzol^® reagent (Beyotime Institute of Biotechnology). Total RNA was quantified at 260 nm with a spectrophotometer, and the purity was evaluated using the ratio of readings at 260 and 280 nm. A PrimeScript RT Reagent kit (Takara Biotechnology Co., Ltd.) was used to synthesize cDNA at 42°C for 45 min. RT-qPCR was conducted using a SYBR ExScript qRT-PCR kit (Takara Biotechnology Co., Ltd.). The reaction procedures were as follows: Initial denaturation at 95°C for 10 min; followed by 38 cycles of 95°C for 30 sec, annealing at 58°C for 30 sec, elongation at 72°C for 1 min; and a final extension at 72°C for 5 min. Primer sequences acquired from Sangon Biotech Co., Ltd., are listed in Table II. The relative expression levels of DANCR, miR-135b-5p and KLF9 were quantified according to the 2^−ΔΔCq calculation (33) and normalized to GAPDH (for DANCR and KLF9) or U6 (for miR-135b-5p).

Human normal plasma cells (nPCs) were purchased from Hunan Fenghui Biotechnology Co., Ltd., and MM cells (RPMI-8226, H929, U266 and MM1S cells) were purchased from the American Type Culture Collection. All cells were cultured in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and penicillin-streptomycin mixed solution (1%). Cultures were incubated at 37°C in a humidified atmosphere with 5% CO₂.

The overexpression vectors of DANCR (pcDNA-DANCR), pcDNA-KLF9 and pcDNA-negative control (NC) were provided by Hanbio Biotechnology Co., Ltd. Short hairpin RNA (sh)-KLF9 and sh-NC were purchased from Guangzhou RiboBio Co., Ltd. miR-135b-5p inhibitor (5′-UCACAUAGGAAUGAAAAGCCAUA-3′), inhibitor NC (5′-UUCAUCGUGUUAUUAGCGUUCCU-3′), miR-135b-5p mimics (5′-UAUGGCUUUUCAUUCCUAUGUGA-3′) and miR-NC (5′-UAUAUCGUGUUAUUAGCGUUCCU-3′) were purchased from Shanghai GenePharma Co., Ltd. The transcripts (20 nM) were transfected into MM cells using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). Transfection was performed for 48 h at 37°C. Subsequently, 48 h after transfection, the transfected cells were harvested to perform further experiments.

Cell viability was measured using the MTT assay. Briefly, RPMI-8226 and H929 cells were seeded in 96-well plates (5×10³ cells/well). Following incubation for 24, 48, 72 and 96 h at 37°C, MTT (20 µl; Sigma-Aldrich; Merck KGaA) was pipetted into each well and the reaction mixture was incubated at 37°C for 4 h. Thereafter, 100 µl dimethyl sulfoxide (200 µl/well; Sigma-Aldrich; Merck KGaA) was added to dissolve the formazan. To determine the cell viability, the optical density value was monitored at 490 nm using a microplate reader (Bio-Rad Laboratories, Inc.).

The 5-ethynyl-2′-deoxyuridine (EdU) assay kit (cat. no. C10310-1; Guangzhou RiboBio Co., Ltd.) was used to measure the number of aforementioned cells undergoing DNA replication, as the thymidine analogue EdU is incorporated into DNA during DNA replication. Briefly, cells were first cultured with 50 µM EdU for 2 h at 37°C, followed by fixing with 4% formaldehyde for 20 min at room temperature, permeabilization with 0.5% Triton X-100 for 20 min and incubation with 1X Apollo reaction cocktail for 30 min at room temperature. Following which, cell nuclei were stained with DAPI (1 µg/ml; 10 min; Sigma-Aldrich; Merck KGaA) at room temperature for 2 h. EdU-positive cells were determined using fluorescence microscopy (magnification, ×400).

The cell invasive ability was assessed using Transwell chambers precoated with Matrigel (8-µm pore size; Corning, Inc.) overnight at 37°C. Briefly, cells (1×10⁵) resuspended in serum-free medium were inoculated into the upper chamber, accompanied by the addition of RPMI-1640 medium containing 10% FBS to the lower chamber. After incubation for 48 h at 37°C, non-invaded cells were wiped off. Invaded cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet for 15 min at 37°C. Finally, the stained cells were analyzed using an inverted light microscope (Olympus Corporation; magnification, ×400).

For the assessment of cell migration, the operation was similar to the cell invasion assay, but the Transwell chambers were not pre-coated with Matrigel.

The miRNA targets of DANCR were predicted using starBase software version 2.0 (http://starbase.sysu.edu.cn/), and 53 targets were predicted. Among these miRNA targets, miR-135b-5p was selected for the following assays due to its important role in MM (25,26) and its unknown regulatory relationship. In addition, the mRNA targets of miR-135b-5p were also predicted using starBase software. In total, 3,791 targets were predicted. KLF9 was selected for the following assays due to its important role in MM (31) and its unknown relationship with miR-135b-5p in MM.

The 3′-untranslated region (UTR) fragment of DANCR or KLF9, including the assumed binding sites for miR-135b-5p, was introduced into psiCHECK2 (Promega Corporation), and named as DANCR wild-type (wt) or KLF9 wt. Similarly, the 3′-UTR portion of DANCR or KLF9 harboring the mutated complementary sites for the miR-135b-5p seed region were inserted into psiCHECK2 (Promega Corporation), and named DANCR mutant (mut) or KLF9 mut. Subsequently, the aforementioned vectors (80 ng), along with the miR-135b-5p mimics or miR-NC (50 nM), were incubated with RPMI-8226 and H929 cells using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 48 h at 37°C. The luciferase activity of cell lysates was examined using a DLR Assay system (Promega Corporation). Relative luciferase activity was defined as the ratio of firefly luciferase activity/Renilla luciferase activity.

RIPA buffer (Beyotime Institute of Biotechnology) was used to extract the protein from RPMI-8226 and H929 cells. The BCA Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.) was used to measure the protein concentration. Protein samples (30 µg) were separated via 10% SDS-PAGE, and then transferred onto PVDF membranes. After blocking the membranes with 5% skimmed milk for 2 h at room temperature, the membranes were incubated at 4°C overnight with primary antibodies, including anti-KLF9 (1:1,000; cat. no. sc-12996; Santa Cruz Biotechnology, Inc.) and anti-GAPDH (1:1,000; cat. no. ab8245; Abcam). Then, the membranes were washed with TBS containing 0.1% Tween-20. The secondary antibody (1:2,000; cat. no. ab205723; Abcam) was then added and the membranes were incubated with the protein samples at 37°C for 1 h. The immunoreactive signals were visualized using an ECL system (BD Biosciences), and the relative protein expression level of KLF9 was analyzed using Alpha Innotech imaging software version 3.1.2 (ProteinSimple).

All experiments were conducted in triplicate in at least three independent experiments. Experimental data were analyzed using SPSS 22.0 software (IBM Corp.) and are presented as the mean ± SD. Differences between two groups were compared using an unpaired Student's t-test. Comparisons between multiple groups were assessed by one-way ANOVA, which was then followed by the Tukey's post hoc test. A χ² test was used for analysis the data in Table I. Correlations among DANCR, miR-135b-5p and KLF9 were assessed using Pearson correlation analysis. P<0.05 was considered to indicate a statistically significant difference.

The expression level of lncRNA DANCR was determined using RT-qPCR. The relative expression level of DANCR was lower in serum samples from patients with MM compared with that in serum samples from healthy donors (P<0.01; Fig. 1A). Moreover, DANCR expression was consistently decreased in the MM cell lines (RPMI-8226, U266, MM1S and H929 cells) compared with that in the nPCs (all P<0.01; Fig. 1B), especially in RPMI-8226 and H929 cells. Therefore, RPMI-8226 and H929 cells were chosen for further experiments.

The association between DANCR and the clinicopathological characteristics of MM cases was also studied, and it was found that DANCR expression was associated with the International Staging System (ISS) stage (P=0.002; Table I).

To evaluate the role of lncRNA DANCR in MM cells, DANCR was initially overexpressed by transfection with pcDNA-DANCR. The addition of pcDNA-DANCR led to a significant increase in the expression level of DANCR compared with the addition of pcDNA-NC in RPMI-8226 and H929 cells (all P<0.01; Fig. 2A). Functional experiments were also performed. The results of the MTT assay indicated that overexpression of DANCR significantly reduced cell viability at 96 h of incubation compared with the pcDNA-NC group in RPMI-8226 and H929 cells (all P<0.01; Fig. 2B). Moreover, the number of RPMI-8226 or H929 cells incorporating EdU in the pcDNA-DANCR group was significantly lower compared with that in the pcDNA-NC group (all P<0.01; Fig. 2C). The results of the Transwell assay also demonstrated that the relative migration and invasion of RPMI-8226 and H929 cells were decreased by overexpression of DANCR (all P<0.01; Fig. 2D and E).

To examine the downstream mechanism of lncRNA DANCR in RPMI-8226 and H929 cells, bioinformatics analysis was performed using starBase and it was shown that DANCR had combinative sites for miR-135b-5p (Fig. 3A), indicating that miR-135b-5p was the target of DANCR. Accordingly, the expression level of miR-135b-5p was determined and the association between DANCR and miR-135b-5p in MM cells was investigated. It was identified that miR-135b-5p was upregulated in the MM cell lines compared with that in nPCs (all P<0.01; Fig. 3B). Moreover, miR-135b-5p expression was decreased by the overexpression of DANCR in RPMI-8226 and H929 cells (all P<0.01; Fig. 3C). Subsequently, a DLR assay was applied to further confirm the cooperation of DANCR and miR-135b-5p, and it was found that the relative luciferase activity of RPMI-8226 and H929 cells co-transfected with DANCR wt and miR-135b-5p mimics was lower compared with that of RPMI-8226 and H929 cells co-transfected with DANCR wt and miR-NC (all P<0.01; Fig. 3D).

It was demonstrated that miR-135b-5p was highly expressed in serum samples of patients with MM as opposed to serum samples from healthy donors (P<0.01; Fig. 3E). There was also a moderate negative correlation between miR-135b-5p expression and DANCR expression in serum samples from patients with MM (P<0.01; Fig. 3F).

The expression of miR-135b-5p was knocked down by the addition of miR-135b-5p inhibitor. The expression level of miR-135b-5p was significantly decreased after the addition of miR-135b-5p inhibitor in RPMI-8226 and H929 cells (all P<0.01; Fig. 4A). The specific effects of miR-135b-5p on RPMI-8226 and H929 cells were then studied. It was found that the knockdown of miR-135b-5p caused a significant decrease in the viability of RPMI-8226 and H929 cells (all P<0.01; Fig. 4B), as well as reduced the migratory and invasive abilities of RPMI-8226 and H929 cells (all P<0.01; Fig. 4C and D).

To elucidate the regulatory mechanism of miR-135b-5p in MM cells, the targets of miR-135b-5p were predicted using starBase and it was found that there were base pairing sites between miR-135b-5p and KLF9 (Fig. 5A). A DLR assay was also performed to corroborate the association between miR-135b-5p and KLF9. It was demonstrated that the addition of miR-135b-5p mimics significantly decreased the relative luciferase activity of the KLF9 wt compared with the addition of miR-NC (all P<0.01; Fig. 5B), but showed no significant effect on the luciferase activity of the KLF9 mut in RPMI-8226 and H929 cells (Fig. 5B). Simultaneously, it was observed that KLF9 expression was significantly downregulated in the MM group compared with the normal group (P<0.01; Fig. 5C). It was found that KLF9 expression was weakly, negatively correlated with miR-135b-5p expression in serum samples from patients with MM (P<0.01; Fig. 5D). In addition, the regulatory association between KLF9 and miR-135b-5p was verified via western blotting, and it was found that the relative protein expression level of KLF9 was downregulated by miR-135b-5p overexpression in RPMI-8226 and H929 cells (all P<0.01; Fig. 5E).

The expression level of KLF9 in MM cell lines was determined. As presented in Fig. 6A, it was found that KLF9 expression was significantly downregulated in MM cell lines compared with that in nPCs (P<0.01). Next, pcDNA-KLF9 or NC was transfected into RPMI-8226 cells to determine transfection efficiency. The results demonstrated that KLF9 expression was significantly increased after pcDNA-KLF9 transfection (P<0.01; Fig. 6B). The effects of KLF9 overexpression on cell viability, migration and invasion were also studied. As shown in Fig. 6C-E, the overexpression of KLF9 significantly repressed the viability, migration and invasion of RPMI-8226 cells (P<0.01).

To determine the association between lncRNA DANCR, KLF9 and miR-135b-5p, KLF9 was silenced and miR-135b-5p was overexpressed in the RPMI-8226 cells. As presented in Fig. 7A and B, the introduction of sh-KLF9 significantly decreased the relative expression level of KLF9, and the introduction of miR-135b-5p mimics elevated the relative expression level of miR-135b-5p in RPMI-8226 cells (all P<0.01). Rescue experiments were then performed. It was demonstrated that the viability of RPMI-8226 cells was reduced in the pcDNA-DANCR group compared with the pcDNA-NC group, whereas this reduction in cell viability mediated by DANCR was partially abrogated by the overexpression of miR-135b-5p or the knockdown of KLF9 in RPMI-8226 cells (all P<0.01; Fig. 7C). Additionally, the relative migration and invasion of RPMI-8226 cells were repressed in the pcDNA-DANCR group compared with the pcDNA-NC group, which was partially abrogated by the overexpression of miR-135b-5p or the knockdown of KLF9 in RPMI-8226 cells (all P<0.01; Fig. 7D and E).