Bone marrow aspirates of 30 untreated II phase MM patients (50-70 years old, half men and half women, serum β-microglobulin=3.5-5.5 mg/l) were collected at the Longgang District People’s Hospital of Shenzhen (Shenzhen, China) from September 2016 to December 2017. Normal bone marrow aspirates were collected from 30 healthy donors with signed statements of informed consent. The bone marrow aspirates were lysed to obtain the bone marrow mononuclear cells (BMMCs), and then the total RNA were extracted by commercial kits for subsequent gene expression analysis. The ethics committee of the Longgang District People’s Hospital of Shenzhen approved the present study.

The RPMI-8266 and U266 human MM cell lines were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in a humidified atmosphere of 95% air and 5% CO₂ at 37°C. The bone mesenchymal stem cells (BMSCs) were isolated from human bone segments, as described previously, and cultured in a standard growth medium for stem cells (28).

The human miR-497 mimic (miR-497) and the negative control (miR-NC) were designed and synthesized by GenePharma (Shanghai, China). When the cells were cultured to 80% confluence, the RNA oligonucleotides were transfected into the cells using Lipo3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. The two miRNAs were used at 50 nM unless otherwise indicated.

For the drug susceptibility assay, bortezomib was dissolved in DMSO at a storage concentration of 10 μM and administered following miRNA transfection for 24 h with a concentration range of 0, 5, 10, 15, and 20 nM when cells reached 90% confluency. Following another 24 h incubation with bortezomib or DMSO at 37°C, the cells were collected for analysis.

An MTT assay was performed for cell viability detection. The cells were seeded into a 96-well plate for culture with serum-free medium for 24 h. Following treatment, the cells were rinsed with PBS and treated with 20 μl of 5 mg/ml MTT for 4 h at 37°C, and DMSO was used to dissolve the formazan precipitation. The absorbance at 570 nm was measured using a plate reader, and the cell viability was calculated as follows: Absorbance (A)=(A_treatemnt−A_control)/A_control×100%.

The cells were rinsed with PBS, trypsinized and collected by centrifugation at 500 × g for 5 min and room temperature (25°C). Following discarding of the supernatant, the cells were resuspended with PBS and rinsed for 3 times, and then ice-cold ethanol was added to fix the cells at −20°C overnight. Following centrifugation (500 × g, 5 min, 25°C) and rinse of 3 times, 1×10⁶ cells were added to a mix of 400 μl RNase (20 μg/ml) together with PI (50 μg/ml) and 0.2% Triton X-100 in PBS, and then incubated for 30 min at 37°C. Finally, the cells were collected, rinsed and centrifuged again (500 × g, 5 min, 25°C), and then resus-pended with PBS for flow cytometric analysis.

For the soft-agar colony formation assay, the cells were mixed with DMEM containing 0.35% low-melting agarose and 10% FBS, and seeded 500 cells onto a coating of 0.7% low-melting agarose in DMEM with 10% FBS. The cells were incubated for up to 3 weeks, following which colonies were visualized by 0.4% crystal violet and imaged using a digital camera (D5600; Nikon Corporation, Tokyo, Japan). Colonies with >0.1 mm diameter were considered as positive.

A TUNEL assay was performed using an in situ Cell Death Detection kit, Fluorescein (Promega Corporation, Madison, WI, USA) according to the manufacturer’s protocol. Images were captured using a fluorescent microscope.

Caspase 3 activity was assayed with a fluorometric kit (R&D Systems, Inc., Minneapolis, MN, USA) following the manufacturer’s protocol. Briefly, the cells were rinsed with PBS and lysed with the lysis buffer from the kit. A substrate of caspase-3 was mixed with the lysate and incubated for 60 min. The fluorescence signal was analyzed at 405 nm.

Total cellular RNA was isolated from the cultured cells using the RNeasy Mini kit (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer’s protocol. RT-qPCR analysis was performed on a 96-well plate ABI Prism 7500 Sequence Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using a 25 μl reaction system containing 12.5 μl 2X SYBR-Green PCR Master mix (Takara Bio, Inc., Otsu, Japan), 100 ng cDNA and 0.4 μM paired primers. The cycling conditions were as follows: 40 cycles of 94°C for 5 sec, 60°C for 34 sec, and 72°C for 30 sec. A comparative 2^−∆∆Cq method (29) was used to calculate the relative expression of target genes. The sequences of Bcl-2 primers were forward, 5′-GGATCCAGGATAACGGAGGC-3′ and reverse, 5′-GATAGGCACCCAGGGTGATG-3′. The relative expression level of Bcl-2 was normalized to the internal reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with primers were forward, 5′-CCAGGTGGTCTCCTCTGA-3′ and reverse, 5′-GCTGTAGCCAAATCGTTGT-3′. The level of mature miR-497 was analyzed by Stem-loop qPCR as described (30) and normalized to that of U6 with primers were forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′.

The protein was extracted with M-PER (Thermo Fisher Scientific, Inc.), quantified using a bicin-choninic acid kit (Thermo Fisher Scientific, Inc.), and 30 μg samples were separated via 12% SDS-PAGE and then transferred onto polyvinylidene difluoride membrane at 100 V for 70 min using a mini transfer tank for electrophoresis (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked in 2% bovine serum albumin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 1 h at room temperature, and incubated with primary antibodies (1:1,000) at 4°C overnight. The membranes were washed in TBS with 0.1% Tween-20 (TBST) three times (5 min each time), and then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (cat. no. ab97051; 1:3,000) for 1 h at room temperature. The blots were analyzed using an enhanced chemiluminescence western blotting detection system (GE Healthcare Life Sciences, Piscataway, NJ, USA). All primary antibodies were obtained from Abcam (Cambridge, UK; BCL-2; cat. no. ab32124; BAX; cat. no. ab32503; caspase-3; cat. no. ab13847; caspase-9; cat. no. ab32539; cytochrome-c; cat. no. ab133504); GAPDH (cat. no. ab9485) was used as the internal reference.

The wild-type 3′ UTR sequence of Bcl-2 containing the predicted miR-497 binding sites using bioinformatics software TargetScan (http://www.targetscan.org) and miRanda (http://cbio.mskcc.org/microrna_data/manual.html) was amplified by PCR using a human cDNA template and paired primers (sense, 5′-GAG TTG CTT TAC GTG GCC TG-3′; antisense, 5′-ACA TGT GTT GGG ATT GCC CT-3′). The corresponding mutated 3′UTR sequence was generated by a TaKaRa MutanBEST kit (Takara Bio, Inc., Otsu, Japan) and primers (mutation-introduced-sense, 5′-CCA ATC CTG TCA GAC GTT CCT GCC AAA AT-3′; antisense, 5′-GTA ACA GTT TCA CTA CTT ATA CCT TAT A-3′) following the manufacturer’s protocol. The two sequences were separately inserted into the pMIR-Reporter vector (Ambion; Thermo Fisher Scientific, Inc.) using restriction enzymes SacI and HindIII. These constructs, in addition to pRL-TK (Promega Corporation) and miR-497/miR-NC, were co-transfected into the cultured cells using Xfect™ Polymer. The luciferase activity was assayed using the Dual-Luciferase^® Reporter (DLR™) Assay system (Promega Corporation) following the manufacturer’s protocol. All samples were assessed in triplicate, and the results were normalized relative to control Renilla activity.

All data values are presented as the mean ± standard deviation from at least three separate determinations. Comparison of all other results was performed by one-way analysis of variance with Tukey’s comparison analysis or Student’s t test, and the statistical significance was analyzed using these tests. P<0.05 was considered to indicate a statistically significant difference. GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA) was used to compare groups.

To elucidate the association between miR-497 and MM, RT-qPCR analysis was performed in 46 MM patients/normal individuals. The expression of miR-497 was lower in patients with MM than in normal individuals (Fig. 1A). Similar experiments were performed in the RPMI-8226 and U266 cell lines, and BMSCs used as a normal control. The results were consistent with the clinical cases, when compared with the normal BMSCs; the level of miR-497 was significantly lower in MM cells (Fig. 1B). Subsequently, the expression of Bcl-2, one of the target genes of miR-497 (20,22,23), detected in MM cells and BMSCs. As expected, it was found that the expression of Bcl-2, at the mRNA level and protein level, was elevated in both MM cell lines (Fig. 1C and D). These results indicated that miR-497 was downregulated but Bcl-2 was upregulated in MM cells, a negative correlation between the two.

It was previously reported that Bcl-2 was closely associated with myeloma cell proliferation (3,5,15). To investigate the effects of miR-497 on myeloma cell proliferation, miR-497 mimic (miR-497) or the matched negative control (miR-NC) were transfected into the RPMI-8266 and U266 MM cell lines to overexpress miR-497 (Fig. 2A). The cell viability and cell cycle were then analyzed through an MTT assay and flow-cytometry, respectively. The results revealed that the overexpression of miR-497 in RPMI-8266 and U266 cells led to decreased cell viability (Fig. 2B) together with altered cell cycle with the majority of cells arrested at the G₀/G₁ phase (Fig. 2C and D).

The soft-agar colony formation assay is another common assay to examine cell proliferation (28,31). In the RPMI-8266 and U266 cells, the number of colonies in the group overex-pressing miR-497 was markedly decreased compared with the miR-NC and control groups (Fig. 2E). Statistical analysis of the colony numbers (Fig. 2F) also indicated that miR-497 disrupted clone formation of MM cells.

Taken together, the overexpression of miR-497 inhibited MM cell proliferation through cell cycle arrest, and reductions in viability and colony formation ability.

It was hypothesized that reduced cell viability may be caused by increased apoptosis (32-34), therefore, subsequent assays of the apoptotic patterns of cells with ectopic miR-497 were performed. Cell apoptosis was analyzed by Annexin V/FACS and TUNEL assays in RPMI-8266 and U266 cells transfected with or without miR-497. The cells with ectopic miR-497 exhibited increased apoptosis (~2.5 fold) according to FACS results (Fig. 3A and B) and TUNEL staining (Fig. 3C). To confirm these findings, the caspase-3 activity was also detected as a common marker for apoptosis (9,31). A consistent result further suggested that the overexpression of miR-497 induced apoptosis by markedly increasing the caspase-3 activity (~2.2 fold) (Fig. 3D). These results indicated that miR-497 promoted MM cell apoptosis.

As a close correlation between Bcl-2 and miR-497 was described in previous reports (20,23), the present study examined whether miR-497 directly targeted the expression of Bcl-2. For this, the Bcl-2 3′UTR and miR-497 sequences were analyzed by bioinformatics software, including TargetScan, miRTarBase, RNAhybrid, and a strong interaction region was found (Fig. 4A). To create an miR-497-insensitive Bcl-2 3′UTR, mutations were introduced into the binding sites (Fig. 4A), and inserted into a luciferase reporter gene plasmid, in addition to the wild-type Bcl-2 3′UTR. A dual-luciferase assay was performed to verify the interaction between miR-497 and Bcl-2. The results revealed that miR-497 significantly decreased the activity of luciferase with the wild-type Bcl-2 3′UTR, but did not affect the activity of luciferase with the mutated Bcl-2 3′UTR (Fig. 4B), suggesting that Bcl-2 serves as a direct target of miR-497, and the interaction site is just as the prediction. To confirm this finding, the present study detected the expression levels of Bcl-2 by RT-qPCR and western blot analyses following miR-497 or miR-NC treatment. The mRNA and protein levels of Bcl-2 were markedly decreased by the overexpression of miR-497, with ~3-fold inhibition (Fig. 4C and D).

Aside from the anti-apoptotic gene Bcl-2, other apoptotic-related proteins also closely associated with Bcl-2 and cell apoptosis. The present study detected the expression of several well-known pro-apoptotic proteins, including Bcl-2-associated X protein (BAX), caspase-3, caspase-9 and cytochrome c (cyto-c) by western blot analysis. All of these proteins were upregulated markedly by the overexpression of miR-497 (Fig. 5A and B).

Bortezomib is a conventional clinical medication in MM therapy; however, bortezomib-resistance can also occur intrinsically or can be acquired in several patients (25). According to the findings of the present study, miR-497 inhibited proliferation and promoted apoptosis of MM cells, indicating that miR-497 may have a potential synergetic effect or sensitization to bortezomib. To confirm this hypothesis, RPMI-8226 and U266 cells, which were have been shown to be more resistant to bortezomib compared with other MM cell lines (26,27), were used. Although the cell viability was decreased when treated with bortezomib treatment alone, a lower level was detected following treatment with ≥10 nM bortezomib combined with miR-497 (Fig. 6A). Subsequently, 10 nM bortezomib treatment for 24 h was selected for the subsequent experiments to analyze cell cycle, colony formation ability and apoptosis. The results showed that the cell cycle arrest was enhanced by the combination of miR-497 and botezomib in the RPMI-8226 and U266 cells (Fig. 6B); the proportion of cells in the G₀/G₁ phase increased from 54.12 to 76.25% when the cells were treated with the addition of miR-497. The colony formation ability was also decreased by the combination of miR-497 and bortezomib, with 50% inhibition compared with bortezomib treatment alone (Fig. 6C). The apoptotic signature analysis by Annexin V/FACS and TUNEL assays demonstrated that miR-497 promoted bortezomib-induced MM cell apoptosis (Fig. 6D and E). As another vital hallmark of apoptosis, the activity of caspase-3 was further detected and, as expected, miR-497 enhanced the activity of caspase-3 when co-treated with bortezomib (Fig. 6F). Taken together, these results led to the conclusion that miR-497 enhanced the chemosensitivity of MM cells to bortezomib, and may serve as a potential therapeutic candidate in combination with bortezomib.