Clin Exp Med DOI 10.1007/s10238-015-0355-4
ORIGINAL ARTICLE
miRNA-202 in bone marrow stromal cells affects the growth and adhesion of multiple myeloma cells by regulating B cell-activating factor Xianjuan Shen1 • Yuehua Guo2 • Jiajia Yu3 • Jing Qi1 • Wei Shi1 • Xinhua Wu1 Hongbing Ni4 • Shaoqing Ju1,2,4
•
Received: 19 December 2014 / Accepted: 24 April 2015 Ó Springer-Verlag Italia 2015
Abstract Bone marrow stromal cells (BMSCs) up-regulate B cell-activating factor (BAFF) in multiple myeloma. Increasing experimental evidence has shown that microRNAs play a causal role in hematology tumorigenesis. In this study, we characterized the role of miR-202 in regulating the expression of BAFF in BMSCs. It was found that expressions of BAFF mRNA and protein were increased in BMSCs treated with miR-202 inhibitor. The growth rate of miR-202 mimics transfection cells was significantly lower than that of non-transfected cells. The expression of Bcl-2 protein was down-regulated, and Bax protein was up-regulated after miR-202 mimics transfection. Over-expression of miR-202 in BMSCs rendered MM cells more sensitive to bortezomib. More significantly, the regulatory effect of miR-202 could inhibit the activation of NF-jB pathway in BMSCs. These results suggest that miR202 functions as a modulator that can negatively regulate BAFF by inhibiting MM cell survival, growth, and adhesion in the bone marrow microenvironment.
Keywords Multiple myeloma (MM) Bone marrow stromal cells (BMSCs) Drug resistance microRNA-202 (miR-202) B cell-activating factor (BAFF) Abbreviations miRNA microRNA BMSCs Bone marrow stromal cells MM Multiple myeloma BAFF B cell-activating factor Bort Bortezomib Thal Thalidomide Dex Dexamethasone TNF Tumor necrosis factors PBMCs Peripheral blood mononuclear cells CAM-DR Cell adhesion-mediated drug resistance ICAM-1 Intercellular adhesion molecule-1 VCAM-1 Vascular cell adhesion molecule-1 VEGF Vascular endothelial growth factor IL-6 Interleukin 6 ELISA Enzyme-linked immunosorbent assay
& Shaoqing Ju [email protected] 1
Surgical Comprehensive Laboratory, Affiliated Hospital of Nantong University, #20 Xisi Road, Nantong 226001, JS, People’s Republic of China
2
Nantong University, #9 Seyuan Road, Nantong 226001, JS, People’s Republic of China
3
The First Hospital Affiliated to Soochow University, #20 Shizi Road, Suzhou 215006, JS, People’s Republic of China
4
Laboratory Medicine Center, Affiliated Hospital of Nantong University, #20 Xisi Road, Nantong 226001, JS, People’s Republic of China
Introduction Multiple myeloma (MM) is an incurable malignant disorder characterized by accumulation of malignant plasma cells within the bone marrow (BM), where stromal cells play an essential role in MM progression through direct and indirect interactions with MM cells to promote MM cell growth, survival, migration, and drug resistance by secreting various factors and cytokines [1, 2]. Of them, bone marrow stromal cells (BMSCs) secrete interleukin 6
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(IL-6), B cell-activating factor (BAFF), and other cytokines to promote MM cell proliferation and drug resistance [3–5]. BAFF, also known as B lymphocyte stimulator, is a member of the tumor necrosis factors (TNF) superfamily and has been identified as a critical factor influencing the growth and survival of malignant B cells [6, 7]. Our previous and other studies [8, 9] found that the expression levels of BAFF and its receptors in serum of MM patients and MM cell line were significantly higher than those in the normal control group. Interestingly, BMSCs secrete at least threefold higher levels of BAFF than MM cells to promote interaction and adhesion of MM cells with BMSCs [10]. Importantly, adhesion-induced NF-jB activation in BMSCs in turn up-regulates BAFF gene expression, and BAFF promotes MM cell growth and protects MM cell against drug-induced cell death [11, 12]. So, BAFF from BMSCs plays a very important biological role in the pathogenesis of MM within the bone marrow microenvironment. Although several experimental studies [13–15] suggested that cytokines and signaling pathways were involved in regulating the expression of BAFF, little is known about the effect of microRNAs (miRNAs) on the regulation of BAFF. A group of tumor-associated miRNAs including miR-181, miR-21, miR-17-92, miR-93, and miR202 were recently profiled in MM patient samples and cell lines using microarray analysis [16, 17]. Our preliminary studies also found that miR-202 was expressed in the peripheral blood mononuclear cells and bone marrow of MM patients. Bioinformatics analysis showed that miR-202 was able to regulate the expression of BAFF. However, the functional activity of miR-202 in MM has not been elucidated. In this study, we demonstrated that BAFF was a miR202 regulation target. miR-202 could inhibit MM cell survival, growth, and adhesion in the bone marrow microenvironment. In addition, over-expression of miR-202 in BMSCs sensitized MM cells to bortezomib. The regulatory role of miR-202 could inhibit the activation of the NF-jB pathway and cell adhesion-mediated drug resistance (CAM-DR).
Materials and methods Materials Materials used in this study were as follows: PsiCHECK-2 vector and luciferase assay system (Promega, Beijing, China); NF-jB p52 and p65 antibodies (BioLegend, San Diego, CA); BAFF monoclonal antibody (R&D Systems, Minneapolis, MN); Bax antibody, Bcl-2 antibody, and b-
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actin antibody (Cell signaling, US); cell proliferation reagent WST-1 (Roche, Germany); Lipofectamine 2000 and reverse transcription reagent (Fermentas, Lithuania); fetal bovine serum (FBS) (Hyclone, USA); DMEM medium (Gibco, USA); siBAFF (BAFF-homo-708) (GenePharma, Shanghai, China); miR-202 mimics, miR202 inhibitor, negative control, and inhibitor negative controls (Invitrogen, Shanghai, China); and bortezomib, thalidomide, and dexamethasone (qcbio Science, Shanghai, China). Patient samples Patients who were clinically diagnosed with MM were admitted to the affiliated hospital of Nantong University (Nantong, China) between August 2012 and December 2013, including three females and four males ranging in age from 50 to 65 years. All the included patients were newly diagnosed and non-treated. CD138? cells from bone marrow aspirates were isolated with a BD FACSAria II using a phycoerythrin (PE)-conjugated anti-CD138 (BD Biosciences, NJ, USA) antibody. BMSCs were obtained from CD138-non-expressing fraction separated from CD138? patient MM cells. All samples were anonymous, and the study protocol was approved by the local ethics committee. Cell culture CD138? cells and BMSCs were cultured in DMEM medium with 20 % FBS. 293T cells, U266 cells (MM cell line), and HS-5 cells (BMSCs cell line) were cultured in DMEM medium with 10 % FBS. For mixed co-culture, U266 cells (4 9 105 cells/well) were serum-starved for 8 h and then directly cultured with HS-5 cells (6 9 104 cells/ well) in 12-well plates. HS-5 cells were transfected with miR-202 mimics, mimic NC, miR-202 inhibitor NC, miR202 inhibitor, and blank control for 12 h and then added myeloma cells U266 co-cultured for 24 h. SYBR Green I real-time PCR Real-time PCR was performed in triplicate with Fast Start Universal SYBR Green Master (Rox) mix kit. BAFF primer sequences were 50 -TGT CAC CGC GGG ACT GAA AAT CT-30 (forward primer) and 50 -TGT CTG CAA TCA GTT GCA AGC AGT-30 (reverse primer), which yielded products of 137 bp. GAPDH primer sequences were 50 TGC TGT TCT GAC TGG AGT TG-30 (forward primer) and 50 -GCT GTC TTG CTG CCT CAC-30 (reverse primer), which yielded products of 172 bp. The miR-202 and U6 primers were purchased from RiboBio Co., Ltd (Guangzhou, China). Each reaction was performed in a
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final volume of 20 ll containing 10 ll SYBR Green I mix (Rox), 3 ll cDNA, 0.5 ll forward primer, 0.5 ll reverse primer, and RNase-free H2O. The mix was incubated at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 31 s. Cell transfection HS-5 cells were seeded in six-well plates at a density of 2–8 9 105/well in 2 ml RPMI-1640 with 10 % FBS, incubated at 37 °C for 12 h, and then transfected with 50 nM miR-202 mimics (50 -AGA GGU AUA GGG CAU GGG AA-30 , 50 -CCC AUG CCC UAU ACC UCU UU-30 ), 50 nM negative control (50 -UUC UCC GAA CGU GUC ACG UTT-30 , 50 -ACG UGA CAC GUU CGG AGA ATT-30 ), 100 nM miR-202 inhibitor (50 -UUC CCA UGC CCU AUA CCU CU-30 ), 100 nM inhibitor negative control (50 -CAG UAC UUU UGU GUA GUA CAA-30 ), and 40 nM siBAFF (50 -CAU GGC UUC UCA GCU UUA ATT-30 , 50 -UUA AAG CUG AGA AGC CAU GTT-30 ), using Lipofectamine 2000 according to the manufacturer’s instructions. Untransfected cells were used as control. After 48–72 h, total RNA and protein were extracted. Luciferase reporter assay Luciferase reporter constructs contained the BAFF 30 UTR or BAFF 30 UTR mutant variant prepared using a psiCHECK-2 vector. Reporter constructs were transfected into 293T cells with miR-202 mimics by Lipofectamine 2000. After 48 h, cells were harvested and analyzed using a dual luciferase reporter assay kit according to the manufacturer’s instructions. Immunoblot analysis Nuclear and cytosolic extracts were prepared according to the manufacturer’s instruction (Pirece Biotechnology, IL, USA). Total protein was extracted using RIPA lysate containing 1 % PMSF. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 100 lg total protein (80 vs. 40 min; 100 vs. 60 min), and the protein was transferred from SDS-PAGE to the PVDF membrane (300 mA 120 min). After blocking with 5 % nonfat milk in Tris-buffered saline containing 0.1 % Tween 20 (TBST), the polyvinylidene fluoride membrane was incubated with primary antibody (Bax, Bcl-2, BAFF, p65, p52, Lamin B, and b-actin) in 5 % bovine serum albumin in TBST overnight at 4 °C, washed three times with TBST, and incubated with secondary antibody in 5 % milk in TBST. After three washes with TBST, the membrane was developed with enhanced chemiluminescence (ECL, Amersham Pharmacia).
WST-1 cell proliferation assay U266 cells were seeded in a 96-well plate at a density of 3000 cells per well. The total volume in each well was 100 ll. Drug concentrations were 10 nM Dex, 200 lM Thal, and 50 nM Bort according to the reference literature [18, 19]. To each well, 10 ll WST-1 reagent was added and incubated for additional 2 h in a 37 °C/5 % CO2 incubator. Absorbance (A) value of each well was measured at 450 nm (650 nm as reference). Five repeated wells were designed for each group, and the experiment was repeated in triplicates. Cell cycle analysis Cell cycle analysis was carried out by FACScan flow cytometry using a standard protocol. Briefly, cells were harvested by centrifugation, washed with PBS, and fixed with cold 70 % ethanol overnight. The fixed cells were then stained with PI solution consisting of 50 lg/ml PI, 20 lg/ ml RNase A, and 0.1 % Triton X-100. After 1-h incubation in the dark, fluorescence-activated cells was sorted in a FACScan flow cytometer. The distribution of cells in the different cell cycle phases was analyzed with a FACSCalibur flow cytometer using CellQuest Pro software. The percentage of cells in each phase of the cell cycle was determined at least in triplicates and expressed as mean ± SD. ELISA Soluble intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), vascular endothelial growth factor (VEGF), and interleukin 6 (IL6) levels were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (NeoBioscience Technology Co., Ltd, Shanghai, China). Optical densities were measured at 450 nm (Spectra II), and concentrations were determined based on the OD values of standard curves. Every parameter was measured five times, and the results were averaged. TUNEL assay of apoptotic cells TdT-mediated dUTP nick end labeling (TUNEL) assay was performed with One Step TUNEL Apoptosis Kit (Beyotime, JS, China). U266 cells (2 9 105) were collected and washed with PBS two times. The cell suspension was put onto poly-L-lysine-coated glass slides, fixed, permeabilized, and incubated with TUNEL reaction mixture at 37 °C for 1 h as described in the manufacturer’s protocol. The cells were analyzed by fluorescence microscopy.
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Statistical analysis Data were analyzed using SPSS 13.0 and are presented as the mean ± SD. The statistical significance of differences between experimental and control groups was determined by Student’s t test: P \ 0.05 was considered to be statistically significant.
Results Expression of BAFF and miR-202 in BMSCs of MM patients According to MM miR signature [20] and recent studies published by other groups using human miRNA targets, mioRNA.org, and DIANA-miT bioinformatics software, we found that miR-202 seemed to be an interesting candidate for BAFF. We first analyzed the expression of BAFF and miR-202 in BMSCs. It was found that the expression of BAFF mRNA was higher in MM-BMSCs and HS-5 cells than that in normal BMSCs as shown by real-time PCR (Fig. 1a). On the other hand, miR-202 was weakly expressed in MM-BMSCs and HS-5 cells (Fig. 1b). Therefore, we further studied the role of miR-202 and BAFF in BMSCs and their effects on MM cells in the subsequent work.
miR-202 mimics, and luciferase activity was measured 48 h after transfection. It was found that the luciferase activity decreased by 39.3 % in 293T cells when BAFF 30 UTR reporter constructs were co-transfected with miR-202 mimics, compared with that of the BAFF 30 -UTR reporter alone (Fig. 2b). Real-time PCR and Western blot were performed to analyze BAFF expression in BMSCs transfected with miR-202 inhibitor or mimics. The results showed that BAFF mRNA was reduced in miR-202 mimics transfection group, but the change in miR-202 inhibitor transfection group was insignificant (Fig. 2c, d). BAFF protein expression was increased in BMSCs and HS-5 cells treated with miR-202 inhibitor as compared with that of untreated and mimics-treated cells, and the change in miR202 mimics transfection group was also significant (Fig. 2e). These results suggest that miR-202 functioned as a modulator of BAFF expression. miR-202 inhibits MM cell survival and growth in the bone marrow microenvironment
We performed a luciferase reporter assay to see whether there was a direct interaction between miR-202 and BAFF 30 -UTR. The result of bioinformatics analysis showed that the miR-202 seed region bounds to the BAFF 30 -UTR (Fig. 2a). Then, we cloned the BAFF 30 -UTR into a psiCHECK-2 dual luciferase reporter construct. Reporter constructs were transfected into 293T cells with or without
HS-5 cells were transfected with miR-202 mimics, mimic NC, miR-202 inhibitor NC, miR-202 inhibitor, and blank control for 12 h and then co-cultured for 24 h after addition of myeloma cells U266. U266 cells were harvested and used in the subsequent experiment. WST-1 assay showed that the growth of miR-202 mimics transfection cells was significantly lower than that of the non-transfected cells at 48 h (P \ 0.05) (Fig. 3a). Meanwhile, as shown in Fig. 3b, there were significantly more apoptotic (green fluorescent) cells after transfection with miR-202 mimics as compared with the controls and miR-202 inhibitor transfection group by TUNEL assays. Western blot analysis also showed that the expression of Bcl-2 protein was down-regulated by approximately 78 %, and the expression of Bax protein was up-regulated by approximately 46 % after transfection with miR-202 mimics (Fig. 3c). Taken together, these
Fig. 1 BAFF and miR-202 expressions in BMSCs from MM patients. a The expressions of BAFF in HS-5 cells and BMSCs from MM patients and normal donors were detected by real-time PCR (P1–
P7 as MM patients; *P \ 0.05). b The expression of miR-202 was detected by real-time PCR in HS-5 cells and BMSCs from MM patients and normal donors (P1–P7 as MM patients; *P \ 0.05)
miR-202 modulates BAFF expression in BMSCs
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Fig. 2 miR-202 modulates BAFF expression in BMSCs. a miR-202 target site located in the 30 UTR of BAFF mRNA is highly conserved in humans. b 293T cells were transfected with miR-202 mimics and psiCHECK-2 constructs (wild-type and mutant BAFF 30 UTRs) simultaneously (*P \ 0.05). c Real-time PCR was used to analyze the expression of BAFF in HS-5 cells transfected with miR-202 mimics NC, miR-202 mimics, inhibitor NC, inhibitor, and si-BAFF as
compared with that in untransfected cells (*P \ 0.05). d 2 % agarose gel electrophoresis was performed on miR-202 real-time PCR product of transfected or untransfected cells. The result showed that the size of BAFF product was 137 bp versus 172 bp of GAPDH product. e Western blot analysis of BAFF in HS-5 cells and BMSCs that transfected with miR-202 mimics, inhibitor, or controls as compared with that in untransfected cells
results demonstrate that miR-202 could decrease the expression of BAFF and inhibit MM cell survival and growth in the bone marrow microenvironment.
vs. 1.290 ± 0.067 ng/ml; P \ 0.05), and it was up-regulated significantly in miR-202 inhibitor group as compared with that in the control group (78.65 ± 5.464 pg/ml vs. 1.690 ± 0.040 ng/ml; P \ 0.05) (Fig. 4a, b). IL-6 and VEGF are critical growth factors for myeloma cells and both are mainly produced by BMSCs [22]; we also detected VEGF and IL-6 secretion in HS-5 cells transfected with miR-202 mimics, miR-202 inhibitor, and blank control. ELISA results demonstrated that the levels of VEGF and IL-6 secreted in the supernatant of HS-5 cells transfected with miR-202 mimics were significantly lower than that of the control group (40.42 ± 3.160 and 14.65 ± 2.649 vs. 70.78 ± 5.582 and 20.85 ± 3.275 pg/ ml; both P \ 0.05) and up-regulated significantly in miR202 inhibitor group (108.6 ± 11.06 and 28.10 ± 2.787 pg/ ml; P \ 0.05) (Fig. 4c, d). Taken together, these data suggest that the expression of adhesion molecules was low in BMSCs transfected with miR-202 mimics, thus indirectly inhibiting their adhesion to MM cells and reducing their protective effect on MM cells.
The effect of miR-202 on MM cell adhesion to BMSCs ICAM-1 (CD54) and VCAM-1 (CD106) are the most important adhesion molecules that mediate cell-to-cell contact between BMSCs and MM cells [21]. To gain insight into how miR-202 affects MM cell survival, we evaluated the expression of adhesion molecules in HS-5 cells transfected with miR-202 mimics, mimic NC, miR-202 inhibitor NC, miR-202 inhibitor, and blank control. The levels of ICAM-1 and VCAM-1 secreted in the supernatant of HS-5 cells were detected by ELISA analysis. It was found that the expression of VCAM-1 and ICAM-1 in the supernatant of BMSCs transfected with miR-202 mimics was significantly lower (49.51 ± 6.331 pg/ml and 1.200 ± 0.079 ng/ml, respectively) than that of the control group (59.24 ± 5.416 pg/ml
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miR-202 up-regulations are associated with apoptosis in response to anti-myeloma drugs
Fig. 3 miR-202 inhibits MM cell survival and growth in the bone marrow microenvironment. HS-5 cells were transfected with miR-202 mimics NC, miR-202 mimics, inhibitor NC, inhibitor si-BAFF, and blank control for 12 h and then co-cultured with myeloma cell U266 for 24 h. U266 cells were harvested for use in the subsequent experiment. a WST-1 assay was used to detect the proliferation of U266 cells (compared with control group, *P \ 0.05). Data were representative of five independent experiments. b TUNEL analysis of U266 cell apoptosis. Free DNA fragments in apoptotic cells were labeled with green fluorescence. c Western blot analysis of the expression of Bcl-2 and Bax. Bcl-2 and Bax levels were normalized to those of b-actin
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HS-5 cells were transfected with miR-202 mimics, mimic NC, or blank control for 12 h and then co-cultured for 24 h after addition of myeloma cells U266. Bortezomib (Bort; 50 nM), thalidomide (Thal; 200 lM), and dexamethasone (Dex; 10 nM) were added to continue the culture for 24 h. U266 cells were harvested and used in the subsequent experiment. Cell cycle distribution was detected by flow cytometry. It was found that combination treatment of Bort and miR-202 mimics triggered an accumulation of cells in the G0/G1 stage, whereas the numbers of cells in the S phases decreased (Table 1). Compared with Bort-alone group, there were significant differences in the numbers of cells in G0/G1 and S phases (61.03 ± 2.74 vs. 52.01 ± 3.66 %, P \ 0.05; 9.11 ± 0.57 vs. 18.29 ± 3.39 %, P \ 0.05). However, there was no significant difference in G0/G1 and S phases between Dex and miR-202 mimics combination treatment group and Dexalone group (50.37 ± 3.52 vs. 58.21 ± 1.81, P [ 0.05; 9.37 ± 1.07 vs. 8.60 ± 1.69, P [ 0.05). The stages of cell cycle in U266 cells treated with combined Thal and miR202 mimics were not significantly different from those in Thal alone group, suggesting that miR-202 mimics decreased the expression of BAFF and then increased the sensitivity of MM cells to Bort significantly but to Dex and Thal insignificantly in the bone marrow microenvironment. Bort combined with miR-202 mimics arrested the cell cycle in S phase in MM cells. To confirm the result of flow cytometry, Western blot and TUNEL were performed. As shown in Fig. 5a, combination treatment with Bort and miR-202 mimics increased the pro-apoptotic effect on Bax expression by approximately 236 versus 41.0 % for Bort alone, or 27.0 % for miR-202 inhibitor with Bort. Meanwhile, the expression of Bcl-2 in the combination treatment group of Bort and miR-202 mimics was significantly down-regulated, showing an inhibition rate of 31.0 versus 17.0 % for Bort alone, or 15.5 % for miR-202 inhibitor and Bort combination. TUNEL assay also showed the similar results (Fig. 5b). There were significantly more apoptotic (green fluorescent) cells after combination treatment of Bort and miR-202 mimics as compared with Bort-alone or miR-202 inhibitor and Bort combination treatment. In brief, we demonstrated that up-regulating miR-202 expression could decrease the protective effect of BMSCs on MM cells, sensitizing MM cells to Bort significantly in the bone marrow microenvironment.
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Fig. 4 miR-202 affects the secretion of cell adhesion molecules in BMSCs. HS-5 cells transfected with miR-202 mimics NC, miR-202 mimics, inhibitor NC, miR-202 inhibitor, si-BAFF, and blank control. a The level of VCAM-1 secreted in the supernatant of HS-5 cells was detected by ELISA (*P \ 0.05). b The level of ICAM-1 secreted in
Table 1 Detection of cell cycle distribution by flow cytometry
Groups
the supernatant of HS-5 cells was detected by ELISA (*P \ 0.05). c ELISA was used to analyze VEGF expression in the supernatant of HS-5 cells (*P \ 0.05). d ELISA was used to analyze IL-6 expression in the supernatant of HS-5 cells (*P \ 0.05). Data are from five independent experiments
G0/G1 (%)
S (%)
G2/M (%) 31.11 ± 3.96
Control
48.58 ± 2.52
20.31 ± 1.46
Bort
52.01 ± 3.66#
18.29 ± 3.39
29.70 ± 5.28
Thal Dex
46.84 ± 4.64 58.21 ± 1.81#
20.31 ± 0.97 8.60 ± 1.69#
32.57 ± 4.28 33.20 ± 3.37
Bort ? mimics NC
49.84 ± 5.83
17.56 ± 1.26
28.25 ± 2.38
Thal ? mimics NC
48.59 ± 6.27
22.32 ± 2.47
32.36 ± 1.69
Dex ? mimics NC
52.09 ± 2.14
8.26 ± 1.58
36.48 ± 3.17
Bort ? mimics
61.03 ± 2.74#*
9.11 ± 0.57#*
Thal ? mimics
47.03 ± 3.20
Dex ? mimics
50.37 ± 3.52
21.29 ± 1.95 9.37 ± 1.07#
29.86 ± 2.99 31.71 ± 4.10 40.25 ± 2.75
HS-5 cells were transfected with miR-202 mimics or mimic NC or blank control for 12 h and then cocultured with myeloma cell U266 for 24 h. After addition of 50 nM Bort, 200 lM Thal, or 10 nM Dex, culture was continued for additional 24 h. U266 cells were harvested for use in the subsequent experiment. The percentage of each cell cycle was determined by flow cytometry at least in triplicates and expressed as mean ± SD #
P \ 0.05, compared with control; * P \ 0.05, compared Bort-alone group
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Fig. 5 Synergistic effects of miR-202 mimics with Bort on MM cells. HS-5 cells were transfected with miR-202 mimics, miR-202 mimics NC, miR-202 inhibitor, inhibitor NC, and blank control for 12 h and then co-cultured with myeloma cell U266 for 24 h. After addition of bortezomib (Bort; 50 nM), culture was continued for additional 24 h. U266 cells were harvested for use in the subsequent experiment. a Western blot analysis of the expression of Bcl-2 and Bax. Bcl-2 and Bax levels were normalized to those of b-actin. b TUNEL analysis of U266 cells. Free DNA fragments in apoptotic cells were labeled with green fluorescence
Fig. 6 Effect of miR-202 on NF-jB signaling pathway in BMSCs. Western blot was analyzed with anti-p65 or anti-p52 antibody. The equal loading in each lane was evaluated by stripping the blot and probing it with antibodies specific to b-actin (for cytoplasmic extracts) or Lamin B (for nuclear extracts). HS-5 cells were transfected with miR-202 mimics NC miR-202 mimics, inhibitor NC miR-202 inhibitor, and blank control for 24 h. Translocation of p65 and p52 proteins to the nucleus were observed in HS-5 cells
regulate the expression of BAFF, which in turn further inhibited the activation of NF-jB pathway and CAM-DR.
The effect of miR-202 on MM cell adhesion to BMSCs and CAM-DR via NF-jB pathway
Discussion
Knowing that BAFF-induced MM cell adhesion to BMSCs was mediated via activation of NF-jB pathway in BMSCs [10], we next asked whether miR-202 altered NFjB pathway in BMSCs when HS-5 cells were transfected with mimic NC, miR-202 inhibitor NC, miR-202 mimics, miR-202 inhibitor, or blank control. Western blot showed that miR-202 mimics decreased the translocation of p65 and p52 proteins to the nucleus in HS-5 cells (Fig. 6). Although the change in miR-202 inhibitor transfection group was insignificant, some important differences were observed between miR-202 inhibitor transfection group and miR-202 mimics transfection group, indicating that miR-202 could inhibit NF-jB signaling pathway in BMSCs and that NF-jB pathway was involved in the regulatory effect of BAFF-induced MM cell adhesion to BMSCs. Our results showed that over-expression of miR-202 in HS-5 cells sensitized MM cells to bortezomib. To support the hypothesis that the increased responsiveness to Bort and decreased CAM-DR were due to the ability of miR202 to repress the BAFF-mediated pathway, in Fig. 6, we also found that they were lower than those in control group, suggesting that miR-202 mimics and si-BAFF had a similar effect. These results showed that miR-202 could reversely
The role of miRNAs in hematologic tumorigenesis is largely anecdotal, including MM. Some recent studies [16, 17] profiled miR-202 was in MM patient specimens and cell lines using microarray analysis. Several studies [23, 24] have shown that aberrant miR-202 expression was associated with breast cancer and lung cancer. However, the functional activity of miR-202 in MM has not been elucidated. In this study, we established a luciferase reporter gene and confirmed that miR-202 could regulate BAFF expression by directly binding to its 30 UTR. miR-202 may function as a modulator of BAFF expression to down-regulate BAFF expression. Our study also showed that miR-202 up-regulation conferred cell survival and growth. There was significantly more apoptosis after transfection with miR-202 mimics as compared with the control and miR-202 inhibitor transfection group. These results suggest that BMSCs provide an ideal survival microenvironment for MM cells and that miR-202 could decrease the expression of BAFF in BMSCs and inhibit MM cell survival and growth in the bone marrow microenvironment. Although the initial overall response rate to bortezomib is promising, most patients who initially responded to bortezomib treatment developed resistance to the drug over
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time. About 65 % patients with recurrent or refractory MM did not respond to bortezomib [21, 25]. CAM-DR to a variety of drugs in MM cells by binding BMSCs is regarded as an important obstacle in MM therapy. Our data show that miR-202 over-expression by a specific mimics may be an efficient means of sensitizing myeloma cells to chemotherapeutic drugs. It was found in this study that miR-202 mimics increased the sensitivity of myeloma cells to Bort but less to Dex and Thal in the bone marrow microenvironment. Compared with Bort treatment alone, combined use of Bort and miR-202 mimics repressed myeloma cell survival markedly and arrested the cell cycle in S phase in MM cells. Although, BMSCs could protect MM cells against bortezomib-induced apoptosis, the protective effect provided by BMSCs was abated when BMSCs were transfected with miR-202 mimics and cocultured with myeloma cells. These results suggest that the regulatory mechanism of miR-202 expression may be a promising target for fine-tuning anti-myeloma therapy. Studies [21, 26] have shown that VEGF and IL-6 play a critical role in MM pathogenesis in the bone marrow milieu. Meanwhile, ICAM-1 (CD54) and VCAM-1 (CD106) are the most important adhesion molecules that mediate cell-to-cell contact between BMSCs and MM cells. We found that the levels of ICAM-1, VCAM-1, VEGF, and IL6 secreted in the supernatant of HS-5 cells transfected with miR-202 mimics were significantly lower than those in the control group. They were up-regulated significantly in miR-202 inhibitor group as compared with the control group. Notably, BMSCs transfected with miR-202 mimics had a lower expression of adhesion molecules, which inhibited their adhesion to MM cells, thus reducing their protective effect on MM cells and repressing CAM-DR. MM cell adhesion to BMSCs also triggers the NF-jBdependent transcription and secretion of cytokines such as IL-6 in BMSCs, which further stimulates MM cell growth, survival, CAM-DR, and migration [24]. Our data show that HS-5 cells transfected with miR-202 mimics could inhibit NF-jB signaling pathway, which is believed to be involved in the regulation of BAFF-induced MM myeloma cell adhesion to BMSCs. Knowing that bortezomib can block NFjB signaling pathway; inhibit MM cell growth, survival, and migration; and modulate the MM microenvironment [27], we suppose that the increased responsiveness to Bort is due to the ability of miR-202 to repress the BAFF-mediated pathway, thus counteracting the reactivation of NFjB signaling pathway, which in turn further inhibits CAMDR. In summary, our results suggest that miR-202 functions as a modulator that can negatively regulate BAFF in MM by inhibiting MM cell survival, growth, and adhesion in the bone marrow microenvironment. The regulatory effect of miR-202 can inhibit the activation of NF-jB pathway and
improve the responsiveness of MM cells to Bort as well, probably due to its counteracting effect on the reactivation of NF-jB signaling. This miRNA may prove to be a new biomarker for assessing response to specific therapies for MM. Acknowledgments Grant support: the National Natural Science Foundation of China (81301498; 81271920); Jiangsu Provincial Program for Medical Innovation Teams and Leading Talents (LJ201133); Scientific Research Subject of Jiangsu Province Health Department (H201422), and the six major human resources project of Jiangsu Province (20012-WS-119), Translational Medicine Project of Affiliated Hospital of Nantong University (TDF-zh201407). Conflict of interest
The authors have no conflicts of interests.
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ORIGINAL ARTICLE
miRNA-202 in bone marrow stromal cells affects the growth and adhesion of multiple myeloma cells by regulating B cell-activating factor Xianjuan Shen1 • Yuehua Guo2 • Jiajia Yu3 • Jing Qi1 • Wei Shi1 • Xinhua Wu1 Hongbing Ni4 • Shaoqing Ju1,2,4
•
Received: 19 December 2014 / Accepted: 24 April 2015 Ó Springer-Verlag Italia 2015
Abstract Bone marrow stromal cells (BMSCs) up-regulate B cell-activating factor (BAFF) in multiple myeloma. Increasing experimental evidence has shown that microRNAs play a causal role in hematology tumorigenesis. In this study, we characterized the role of miR-202 in regulating the expression of BAFF in BMSCs. It was found that expressions of BAFF mRNA and protein were increased in BMSCs treated with miR-202 inhibitor. The growth rate of miR-202 mimics transfection cells was significantly lower than that of non-transfected cells. The expression of Bcl-2 protein was down-regulated, and Bax protein was up-regulated after miR-202 mimics transfection. Over-expression of miR-202 in BMSCs rendered MM cells more sensitive to bortezomib. More significantly, the regulatory effect of miR-202 could inhibit the activation of NF-jB pathway in BMSCs. These results suggest that miR202 functions as a modulator that can negatively regulate BAFF by inhibiting MM cell survival, growth, and adhesion in the bone marrow microenvironment.
Keywords Multiple myeloma (MM) Bone marrow stromal cells (BMSCs) Drug resistance microRNA-202 (miR-202) B cell-activating factor (BAFF) Abbreviations miRNA microRNA BMSCs Bone marrow stromal cells MM Multiple myeloma BAFF B cell-activating factor Bort Bortezomib Thal Thalidomide Dex Dexamethasone TNF Tumor necrosis factors PBMCs Peripheral blood mononuclear cells CAM-DR Cell adhesion-mediated drug resistance ICAM-1 Intercellular adhesion molecule-1 VCAM-1 Vascular cell adhesion molecule-1 VEGF Vascular endothelial growth factor IL-6 Interleukin 6 ELISA Enzyme-linked immunosorbent assay
& Shaoqing Ju [email protected] 1
Surgical Comprehensive Laboratory, Affiliated Hospital of Nantong University, #20 Xisi Road, Nantong 226001, JS, People’s Republic of China
2
Nantong University, #9 Seyuan Road, Nantong 226001, JS, People’s Republic of China
3
The First Hospital Affiliated to Soochow University, #20 Shizi Road, Suzhou 215006, JS, People’s Republic of China
4
Laboratory Medicine Center, Affiliated Hospital of Nantong University, #20 Xisi Road, Nantong 226001, JS, People’s Republic of China
Introduction Multiple myeloma (MM) is an incurable malignant disorder characterized by accumulation of malignant plasma cells within the bone marrow (BM), where stromal cells play an essential role in MM progression through direct and indirect interactions with MM cells to promote MM cell growth, survival, migration, and drug resistance by secreting various factors and cytokines [1, 2]. Of them, bone marrow stromal cells (BMSCs) secrete interleukin 6
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(IL-6), B cell-activating factor (BAFF), and other cytokines to promote MM cell proliferation and drug resistance [3–5]. BAFF, also known as B lymphocyte stimulator, is a member of the tumor necrosis factors (TNF) superfamily and has been identified as a critical factor influencing the growth and survival of malignant B cells [6, 7]. Our previous and other studies [8, 9] found that the expression levels of BAFF and its receptors in serum of MM patients and MM cell line were significantly higher than those in the normal control group. Interestingly, BMSCs secrete at least threefold higher levels of BAFF than MM cells to promote interaction and adhesion of MM cells with BMSCs [10]. Importantly, adhesion-induced NF-jB activation in BMSCs in turn up-regulates BAFF gene expression, and BAFF promotes MM cell growth and protects MM cell against drug-induced cell death [11, 12]. So, BAFF from BMSCs plays a very important biological role in the pathogenesis of MM within the bone marrow microenvironment. Although several experimental studies [13–15] suggested that cytokines and signaling pathways were involved in regulating the expression of BAFF, little is known about the effect of microRNAs (miRNAs) on the regulation of BAFF. A group of tumor-associated miRNAs including miR-181, miR-21, miR-17-92, miR-93, and miR202 were recently profiled in MM patient samples and cell lines using microarray analysis [16, 17]. Our preliminary studies also found that miR-202 was expressed in the peripheral blood mononuclear cells and bone marrow of MM patients. Bioinformatics analysis showed that miR-202 was able to regulate the expression of BAFF. However, the functional activity of miR-202 in MM has not been elucidated. In this study, we demonstrated that BAFF was a miR202 regulation target. miR-202 could inhibit MM cell survival, growth, and adhesion in the bone marrow microenvironment. In addition, over-expression of miR-202 in BMSCs sensitized MM cells to bortezomib. The regulatory role of miR-202 could inhibit the activation of the NF-jB pathway and cell adhesion-mediated drug resistance (CAM-DR).
Materials and methods Materials Materials used in this study were as follows: PsiCHECK-2 vector and luciferase assay system (Promega, Beijing, China); NF-jB p52 and p65 antibodies (BioLegend, San Diego, CA); BAFF monoclonal antibody (R&D Systems, Minneapolis, MN); Bax antibody, Bcl-2 antibody, and b-
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actin antibody (Cell signaling, US); cell proliferation reagent WST-1 (Roche, Germany); Lipofectamine 2000 and reverse transcription reagent (Fermentas, Lithuania); fetal bovine serum (FBS) (Hyclone, USA); DMEM medium (Gibco, USA); siBAFF (BAFF-homo-708) (GenePharma, Shanghai, China); miR-202 mimics, miR202 inhibitor, negative control, and inhibitor negative controls (Invitrogen, Shanghai, China); and bortezomib, thalidomide, and dexamethasone (qcbio Science, Shanghai, China). Patient samples Patients who were clinically diagnosed with MM were admitted to the affiliated hospital of Nantong University (Nantong, China) between August 2012 and December 2013, including three females and four males ranging in age from 50 to 65 years. All the included patients were newly diagnosed and non-treated. CD138? cells from bone marrow aspirates were isolated with a BD FACSAria II using a phycoerythrin (PE)-conjugated anti-CD138 (BD Biosciences, NJ, USA) antibody. BMSCs were obtained from CD138-non-expressing fraction separated from CD138? patient MM cells. All samples were anonymous, and the study protocol was approved by the local ethics committee. Cell culture CD138? cells and BMSCs were cultured in DMEM medium with 20 % FBS. 293T cells, U266 cells (MM cell line), and HS-5 cells (BMSCs cell line) were cultured in DMEM medium with 10 % FBS. For mixed co-culture, U266 cells (4 9 105 cells/well) were serum-starved for 8 h and then directly cultured with HS-5 cells (6 9 104 cells/ well) in 12-well plates. HS-5 cells were transfected with miR-202 mimics, mimic NC, miR-202 inhibitor NC, miR202 inhibitor, and blank control for 12 h and then added myeloma cells U266 co-cultured for 24 h. SYBR Green I real-time PCR Real-time PCR was performed in triplicate with Fast Start Universal SYBR Green Master (Rox) mix kit. BAFF primer sequences were 50 -TGT CAC CGC GGG ACT GAA AAT CT-30 (forward primer) and 50 -TGT CTG CAA TCA GTT GCA AGC AGT-30 (reverse primer), which yielded products of 137 bp. GAPDH primer sequences were 50 TGC TGT TCT GAC TGG AGT TG-30 (forward primer) and 50 -GCT GTC TTG CTG CCT CAC-30 (reverse primer), which yielded products of 172 bp. The miR-202 and U6 primers were purchased from RiboBio Co., Ltd (Guangzhou, China). Each reaction was performed in a
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final volume of 20 ll containing 10 ll SYBR Green I mix (Rox), 3 ll cDNA, 0.5 ll forward primer, 0.5 ll reverse primer, and RNase-free H2O. The mix was incubated at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 31 s. Cell transfection HS-5 cells were seeded in six-well plates at a density of 2–8 9 105/well in 2 ml RPMI-1640 with 10 % FBS, incubated at 37 °C for 12 h, and then transfected with 50 nM miR-202 mimics (50 -AGA GGU AUA GGG CAU GGG AA-30 , 50 -CCC AUG CCC UAU ACC UCU UU-30 ), 50 nM negative control (50 -UUC UCC GAA CGU GUC ACG UTT-30 , 50 -ACG UGA CAC GUU CGG AGA ATT-30 ), 100 nM miR-202 inhibitor (50 -UUC CCA UGC CCU AUA CCU CU-30 ), 100 nM inhibitor negative control (50 -CAG UAC UUU UGU GUA GUA CAA-30 ), and 40 nM siBAFF (50 -CAU GGC UUC UCA GCU UUA ATT-30 , 50 -UUA AAG CUG AGA AGC CAU GTT-30 ), using Lipofectamine 2000 according to the manufacturer’s instructions. Untransfected cells were used as control. After 48–72 h, total RNA and protein were extracted. Luciferase reporter assay Luciferase reporter constructs contained the BAFF 30 UTR or BAFF 30 UTR mutant variant prepared using a psiCHECK-2 vector. Reporter constructs were transfected into 293T cells with miR-202 mimics by Lipofectamine 2000. After 48 h, cells were harvested and analyzed using a dual luciferase reporter assay kit according to the manufacturer’s instructions. Immunoblot analysis Nuclear and cytosolic extracts were prepared according to the manufacturer’s instruction (Pirece Biotechnology, IL, USA). Total protein was extracted using RIPA lysate containing 1 % PMSF. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 100 lg total protein (80 vs. 40 min; 100 vs. 60 min), and the protein was transferred from SDS-PAGE to the PVDF membrane (300 mA 120 min). After blocking with 5 % nonfat milk in Tris-buffered saline containing 0.1 % Tween 20 (TBST), the polyvinylidene fluoride membrane was incubated with primary antibody (Bax, Bcl-2, BAFF, p65, p52, Lamin B, and b-actin) in 5 % bovine serum albumin in TBST overnight at 4 °C, washed three times with TBST, and incubated with secondary antibody in 5 % milk in TBST. After three washes with TBST, the membrane was developed with enhanced chemiluminescence (ECL, Amersham Pharmacia).
WST-1 cell proliferation assay U266 cells were seeded in a 96-well plate at a density of 3000 cells per well. The total volume in each well was 100 ll. Drug concentrations were 10 nM Dex, 200 lM Thal, and 50 nM Bort according to the reference literature [18, 19]. To each well, 10 ll WST-1 reagent was added and incubated for additional 2 h in a 37 °C/5 % CO2 incubator. Absorbance (A) value of each well was measured at 450 nm (650 nm as reference). Five repeated wells were designed for each group, and the experiment was repeated in triplicates. Cell cycle analysis Cell cycle analysis was carried out by FACScan flow cytometry using a standard protocol. Briefly, cells were harvested by centrifugation, washed with PBS, and fixed with cold 70 % ethanol overnight. The fixed cells were then stained with PI solution consisting of 50 lg/ml PI, 20 lg/ ml RNase A, and 0.1 % Triton X-100. After 1-h incubation in the dark, fluorescence-activated cells was sorted in a FACScan flow cytometer. The distribution of cells in the different cell cycle phases was analyzed with a FACSCalibur flow cytometer using CellQuest Pro software. The percentage of cells in each phase of the cell cycle was determined at least in triplicates and expressed as mean ± SD. ELISA Soluble intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), vascular endothelial growth factor (VEGF), and interleukin 6 (IL6) levels were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (NeoBioscience Technology Co., Ltd, Shanghai, China). Optical densities were measured at 450 nm (Spectra II), and concentrations were determined based on the OD values of standard curves. Every parameter was measured five times, and the results were averaged. TUNEL assay of apoptotic cells TdT-mediated dUTP nick end labeling (TUNEL) assay was performed with One Step TUNEL Apoptosis Kit (Beyotime, JS, China). U266 cells (2 9 105) were collected and washed with PBS two times. The cell suspension was put onto poly-L-lysine-coated glass slides, fixed, permeabilized, and incubated with TUNEL reaction mixture at 37 °C for 1 h as described in the manufacturer’s protocol. The cells were analyzed by fluorescence microscopy.
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Statistical analysis Data were analyzed using SPSS 13.0 and are presented as the mean ± SD. The statistical significance of differences between experimental and control groups was determined by Student’s t test: P \ 0.05 was considered to be statistically significant.
Results Expression of BAFF and miR-202 in BMSCs of MM patients According to MM miR signature [20] and recent studies published by other groups using human miRNA targets, mioRNA.org, and DIANA-miT bioinformatics software, we found that miR-202 seemed to be an interesting candidate for BAFF. We first analyzed the expression of BAFF and miR-202 in BMSCs. It was found that the expression of BAFF mRNA was higher in MM-BMSCs and HS-5 cells than that in normal BMSCs as shown by real-time PCR (Fig. 1a). On the other hand, miR-202 was weakly expressed in MM-BMSCs and HS-5 cells (Fig. 1b). Therefore, we further studied the role of miR-202 and BAFF in BMSCs and their effects on MM cells in the subsequent work.
miR-202 mimics, and luciferase activity was measured 48 h after transfection. It was found that the luciferase activity decreased by 39.3 % in 293T cells when BAFF 30 UTR reporter constructs were co-transfected with miR-202 mimics, compared with that of the BAFF 30 -UTR reporter alone (Fig. 2b). Real-time PCR and Western blot were performed to analyze BAFF expression in BMSCs transfected with miR-202 inhibitor or mimics. The results showed that BAFF mRNA was reduced in miR-202 mimics transfection group, but the change in miR-202 inhibitor transfection group was insignificant (Fig. 2c, d). BAFF protein expression was increased in BMSCs and HS-5 cells treated with miR-202 inhibitor as compared with that of untreated and mimics-treated cells, and the change in miR202 mimics transfection group was also significant (Fig. 2e). These results suggest that miR-202 functioned as a modulator of BAFF expression. miR-202 inhibits MM cell survival and growth in the bone marrow microenvironment
We performed a luciferase reporter assay to see whether there was a direct interaction between miR-202 and BAFF 30 -UTR. The result of bioinformatics analysis showed that the miR-202 seed region bounds to the BAFF 30 -UTR (Fig. 2a). Then, we cloned the BAFF 30 -UTR into a psiCHECK-2 dual luciferase reporter construct. Reporter constructs were transfected into 293T cells with or without
HS-5 cells were transfected with miR-202 mimics, mimic NC, miR-202 inhibitor NC, miR-202 inhibitor, and blank control for 12 h and then co-cultured for 24 h after addition of myeloma cells U266. U266 cells were harvested and used in the subsequent experiment. WST-1 assay showed that the growth of miR-202 mimics transfection cells was significantly lower than that of the non-transfected cells at 48 h (P \ 0.05) (Fig. 3a). Meanwhile, as shown in Fig. 3b, there were significantly more apoptotic (green fluorescent) cells after transfection with miR-202 mimics as compared with the controls and miR-202 inhibitor transfection group by TUNEL assays. Western blot analysis also showed that the expression of Bcl-2 protein was down-regulated by approximately 78 %, and the expression of Bax protein was up-regulated by approximately 46 % after transfection with miR-202 mimics (Fig. 3c). Taken together, these
Fig. 1 BAFF and miR-202 expressions in BMSCs from MM patients. a The expressions of BAFF in HS-5 cells and BMSCs from MM patients and normal donors were detected by real-time PCR (P1–
P7 as MM patients; *P \ 0.05). b The expression of miR-202 was detected by real-time PCR in HS-5 cells and BMSCs from MM patients and normal donors (P1–P7 as MM patients; *P \ 0.05)
miR-202 modulates BAFF expression in BMSCs
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Fig. 2 miR-202 modulates BAFF expression in BMSCs. a miR-202 target site located in the 30 UTR of BAFF mRNA is highly conserved in humans. b 293T cells were transfected with miR-202 mimics and psiCHECK-2 constructs (wild-type and mutant BAFF 30 UTRs) simultaneously (*P \ 0.05). c Real-time PCR was used to analyze the expression of BAFF in HS-5 cells transfected with miR-202 mimics NC, miR-202 mimics, inhibitor NC, inhibitor, and si-BAFF as
compared with that in untransfected cells (*P \ 0.05). d 2 % agarose gel electrophoresis was performed on miR-202 real-time PCR product of transfected or untransfected cells. The result showed that the size of BAFF product was 137 bp versus 172 bp of GAPDH product. e Western blot analysis of BAFF in HS-5 cells and BMSCs that transfected with miR-202 mimics, inhibitor, or controls as compared with that in untransfected cells
results demonstrate that miR-202 could decrease the expression of BAFF and inhibit MM cell survival and growth in the bone marrow microenvironment.
vs. 1.290 ± 0.067 ng/ml; P \ 0.05), and it was up-regulated significantly in miR-202 inhibitor group as compared with that in the control group (78.65 ± 5.464 pg/ml vs. 1.690 ± 0.040 ng/ml; P \ 0.05) (Fig. 4a, b). IL-6 and VEGF are critical growth factors for myeloma cells and both are mainly produced by BMSCs [22]; we also detected VEGF and IL-6 secretion in HS-5 cells transfected with miR-202 mimics, miR-202 inhibitor, and blank control. ELISA results demonstrated that the levels of VEGF and IL-6 secreted in the supernatant of HS-5 cells transfected with miR-202 mimics were significantly lower than that of the control group (40.42 ± 3.160 and 14.65 ± 2.649 vs. 70.78 ± 5.582 and 20.85 ± 3.275 pg/ ml; both P \ 0.05) and up-regulated significantly in miR202 inhibitor group (108.6 ± 11.06 and 28.10 ± 2.787 pg/ ml; P \ 0.05) (Fig. 4c, d). Taken together, these data suggest that the expression of adhesion molecules was low in BMSCs transfected with miR-202 mimics, thus indirectly inhibiting their adhesion to MM cells and reducing their protective effect on MM cells.
The effect of miR-202 on MM cell adhesion to BMSCs ICAM-1 (CD54) and VCAM-1 (CD106) are the most important adhesion molecules that mediate cell-to-cell contact between BMSCs and MM cells [21]. To gain insight into how miR-202 affects MM cell survival, we evaluated the expression of adhesion molecules in HS-5 cells transfected with miR-202 mimics, mimic NC, miR-202 inhibitor NC, miR-202 inhibitor, and blank control. The levels of ICAM-1 and VCAM-1 secreted in the supernatant of HS-5 cells were detected by ELISA analysis. It was found that the expression of VCAM-1 and ICAM-1 in the supernatant of BMSCs transfected with miR-202 mimics was significantly lower (49.51 ± 6.331 pg/ml and 1.200 ± 0.079 ng/ml, respectively) than that of the control group (59.24 ± 5.416 pg/ml
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miR-202 up-regulations are associated with apoptosis in response to anti-myeloma drugs
Fig. 3 miR-202 inhibits MM cell survival and growth in the bone marrow microenvironment. HS-5 cells were transfected with miR-202 mimics NC, miR-202 mimics, inhibitor NC, inhibitor si-BAFF, and blank control for 12 h and then co-cultured with myeloma cell U266 for 24 h. U266 cells were harvested for use in the subsequent experiment. a WST-1 assay was used to detect the proliferation of U266 cells (compared with control group, *P \ 0.05). Data were representative of five independent experiments. b TUNEL analysis of U266 cell apoptosis. Free DNA fragments in apoptotic cells were labeled with green fluorescence. c Western blot analysis of the expression of Bcl-2 and Bax. Bcl-2 and Bax levels were normalized to those of b-actin
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HS-5 cells were transfected with miR-202 mimics, mimic NC, or blank control for 12 h and then co-cultured for 24 h after addition of myeloma cells U266. Bortezomib (Bort; 50 nM), thalidomide (Thal; 200 lM), and dexamethasone (Dex; 10 nM) were added to continue the culture for 24 h. U266 cells were harvested and used in the subsequent experiment. Cell cycle distribution was detected by flow cytometry. It was found that combination treatment of Bort and miR-202 mimics triggered an accumulation of cells in the G0/G1 stage, whereas the numbers of cells in the S phases decreased (Table 1). Compared with Bort-alone group, there were significant differences in the numbers of cells in G0/G1 and S phases (61.03 ± 2.74 vs. 52.01 ± 3.66 %, P \ 0.05; 9.11 ± 0.57 vs. 18.29 ± 3.39 %, P \ 0.05). However, there was no significant difference in G0/G1 and S phases between Dex and miR-202 mimics combination treatment group and Dexalone group (50.37 ± 3.52 vs. 58.21 ± 1.81, P [ 0.05; 9.37 ± 1.07 vs. 8.60 ± 1.69, P [ 0.05). The stages of cell cycle in U266 cells treated with combined Thal and miR202 mimics were not significantly different from those in Thal alone group, suggesting that miR-202 mimics decreased the expression of BAFF and then increased the sensitivity of MM cells to Bort significantly but to Dex and Thal insignificantly in the bone marrow microenvironment. Bort combined with miR-202 mimics arrested the cell cycle in S phase in MM cells. To confirm the result of flow cytometry, Western blot and TUNEL were performed. As shown in Fig. 5a, combination treatment with Bort and miR-202 mimics increased the pro-apoptotic effect on Bax expression by approximately 236 versus 41.0 % for Bort alone, or 27.0 % for miR-202 inhibitor with Bort. Meanwhile, the expression of Bcl-2 in the combination treatment group of Bort and miR-202 mimics was significantly down-regulated, showing an inhibition rate of 31.0 versus 17.0 % for Bort alone, or 15.5 % for miR-202 inhibitor and Bort combination. TUNEL assay also showed the similar results (Fig. 5b). There were significantly more apoptotic (green fluorescent) cells after combination treatment of Bort and miR-202 mimics as compared with Bort-alone or miR-202 inhibitor and Bort combination treatment. In brief, we demonstrated that up-regulating miR-202 expression could decrease the protective effect of BMSCs on MM cells, sensitizing MM cells to Bort significantly in the bone marrow microenvironment.
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Fig. 4 miR-202 affects the secretion of cell adhesion molecules in BMSCs. HS-5 cells transfected with miR-202 mimics NC, miR-202 mimics, inhibitor NC, miR-202 inhibitor, si-BAFF, and blank control. a The level of VCAM-1 secreted in the supernatant of HS-5 cells was detected by ELISA (*P \ 0.05). b The level of ICAM-1 secreted in
Table 1 Detection of cell cycle distribution by flow cytometry
Groups
the supernatant of HS-5 cells was detected by ELISA (*P \ 0.05). c ELISA was used to analyze VEGF expression in the supernatant of HS-5 cells (*P \ 0.05). d ELISA was used to analyze IL-6 expression in the supernatant of HS-5 cells (*P \ 0.05). Data are from five independent experiments
G0/G1 (%)
S (%)
G2/M (%) 31.11 ± 3.96
Control
48.58 ± 2.52
20.31 ± 1.46
Bort
52.01 ± 3.66#
18.29 ± 3.39
29.70 ± 5.28
Thal Dex
46.84 ± 4.64 58.21 ± 1.81#
20.31 ± 0.97 8.60 ± 1.69#
32.57 ± 4.28 33.20 ± 3.37
Bort ? mimics NC
49.84 ± 5.83
17.56 ± 1.26
28.25 ± 2.38
Thal ? mimics NC
48.59 ± 6.27
22.32 ± 2.47
32.36 ± 1.69
Dex ? mimics NC
52.09 ± 2.14
8.26 ± 1.58
36.48 ± 3.17
Bort ? mimics
61.03 ± 2.74#*
9.11 ± 0.57#*
Thal ? mimics
47.03 ± 3.20
Dex ? mimics
50.37 ± 3.52
21.29 ± 1.95 9.37 ± 1.07#
29.86 ± 2.99 31.71 ± 4.10 40.25 ± 2.75
HS-5 cells were transfected with miR-202 mimics or mimic NC or blank control for 12 h and then cocultured with myeloma cell U266 for 24 h. After addition of 50 nM Bort, 200 lM Thal, or 10 nM Dex, culture was continued for additional 24 h. U266 cells were harvested for use in the subsequent experiment. The percentage of each cell cycle was determined by flow cytometry at least in triplicates and expressed as mean ± SD #
P \ 0.05, compared with control; * P \ 0.05, compared Bort-alone group
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Fig. 5 Synergistic effects of miR-202 mimics with Bort on MM cells. HS-5 cells were transfected with miR-202 mimics, miR-202 mimics NC, miR-202 inhibitor, inhibitor NC, and blank control for 12 h and then co-cultured with myeloma cell U266 for 24 h. After addition of bortezomib (Bort; 50 nM), culture was continued for additional 24 h. U266 cells were harvested for use in the subsequent experiment. a Western blot analysis of the expression of Bcl-2 and Bax. Bcl-2 and Bax levels were normalized to those of b-actin. b TUNEL analysis of U266 cells. Free DNA fragments in apoptotic cells were labeled with green fluorescence
Fig. 6 Effect of miR-202 on NF-jB signaling pathway in BMSCs. Western blot was analyzed with anti-p65 or anti-p52 antibody. The equal loading in each lane was evaluated by stripping the blot and probing it with antibodies specific to b-actin (for cytoplasmic extracts) or Lamin B (for nuclear extracts). HS-5 cells were transfected with miR-202 mimics NC miR-202 mimics, inhibitor NC miR-202 inhibitor, and blank control for 24 h. Translocation of p65 and p52 proteins to the nucleus were observed in HS-5 cells
regulate the expression of BAFF, which in turn further inhibited the activation of NF-jB pathway and CAM-DR.
The effect of miR-202 on MM cell adhesion to BMSCs and CAM-DR via NF-jB pathway
Discussion
Knowing that BAFF-induced MM cell adhesion to BMSCs was mediated via activation of NF-jB pathway in BMSCs [10], we next asked whether miR-202 altered NFjB pathway in BMSCs when HS-5 cells were transfected with mimic NC, miR-202 inhibitor NC, miR-202 mimics, miR-202 inhibitor, or blank control. Western blot showed that miR-202 mimics decreased the translocation of p65 and p52 proteins to the nucleus in HS-5 cells (Fig. 6). Although the change in miR-202 inhibitor transfection group was insignificant, some important differences were observed between miR-202 inhibitor transfection group and miR-202 mimics transfection group, indicating that miR-202 could inhibit NF-jB signaling pathway in BMSCs and that NF-jB pathway was involved in the regulatory effect of BAFF-induced MM cell adhesion to BMSCs. Our results showed that over-expression of miR-202 in HS-5 cells sensitized MM cells to bortezomib. To support the hypothesis that the increased responsiveness to Bort and decreased CAM-DR were due to the ability of miR202 to repress the BAFF-mediated pathway, in Fig. 6, we also found that they were lower than those in control group, suggesting that miR-202 mimics and si-BAFF had a similar effect. These results showed that miR-202 could reversely
The role of miRNAs in hematologic tumorigenesis is largely anecdotal, including MM. Some recent studies [16, 17] profiled miR-202 was in MM patient specimens and cell lines using microarray analysis. Several studies [23, 24] have shown that aberrant miR-202 expression was associated with breast cancer and lung cancer. However, the functional activity of miR-202 in MM has not been elucidated. In this study, we established a luciferase reporter gene and confirmed that miR-202 could regulate BAFF expression by directly binding to its 30 UTR. miR-202 may function as a modulator of BAFF expression to down-regulate BAFF expression. Our study also showed that miR-202 up-regulation conferred cell survival and growth. There was significantly more apoptosis after transfection with miR-202 mimics as compared with the control and miR-202 inhibitor transfection group. These results suggest that BMSCs provide an ideal survival microenvironment for MM cells and that miR-202 could decrease the expression of BAFF in BMSCs and inhibit MM cell survival and growth in the bone marrow microenvironment. Although the initial overall response rate to bortezomib is promising, most patients who initially responded to bortezomib treatment developed resistance to the drug over
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time. About 65 % patients with recurrent or refractory MM did not respond to bortezomib [21, 25]. CAM-DR to a variety of drugs in MM cells by binding BMSCs is regarded as an important obstacle in MM therapy. Our data show that miR-202 over-expression by a specific mimics may be an efficient means of sensitizing myeloma cells to chemotherapeutic drugs. It was found in this study that miR-202 mimics increased the sensitivity of myeloma cells to Bort but less to Dex and Thal in the bone marrow microenvironment. Compared with Bort treatment alone, combined use of Bort and miR-202 mimics repressed myeloma cell survival markedly and arrested the cell cycle in S phase in MM cells. Although, BMSCs could protect MM cells against bortezomib-induced apoptosis, the protective effect provided by BMSCs was abated when BMSCs were transfected with miR-202 mimics and cocultured with myeloma cells. These results suggest that the regulatory mechanism of miR-202 expression may be a promising target for fine-tuning anti-myeloma therapy. Studies [21, 26] have shown that VEGF and IL-6 play a critical role in MM pathogenesis in the bone marrow milieu. Meanwhile, ICAM-1 (CD54) and VCAM-1 (CD106) are the most important adhesion molecules that mediate cell-to-cell contact between BMSCs and MM cells. We found that the levels of ICAM-1, VCAM-1, VEGF, and IL6 secreted in the supernatant of HS-5 cells transfected with miR-202 mimics were significantly lower than those in the control group. They were up-regulated significantly in miR-202 inhibitor group as compared with the control group. Notably, BMSCs transfected with miR-202 mimics had a lower expression of adhesion molecules, which inhibited their adhesion to MM cells, thus reducing their protective effect on MM cells and repressing CAM-DR. MM cell adhesion to BMSCs also triggers the NF-jBdependent transcription and secretion of cytokines such as IL-6 in BMSCs, which further stimulates MM cell growth, survival, CAM-DR, and migration [24]. Our data show that HS-5 cells transfected with miR-202 mimics could inhibit NF-jB signaling pathway, which is believed to be involved in the regulation of BAFF-induced MM myeloma cell adhesion to BMSCs. Knowing that bortezomib can block NFjB signaling pathway; inhibit MM cell growth, survival, and migration; and modulate the MM microenvironment [27], we suppose that the increased responsiveness to Bort is due to the ability of miR-202 to repress the BAFF-mediated pathway, thus counteracting the reactivation of NFjB signaling pathway, which in turn further inhibits CAMDR. In summary, our results suggest that miR-202 functions as a modulator that can negatively regulate BAFF in MM by inhibiting MM cell survival, growth, and adhesion in the bone marrow microenvironment. The regulatory effect of miR-202 can inhibit the activation of NF-jB pathway and
improve the responsiveness of MM cells to Bort as well, probably due to its counteracting effect on the reactivation of NF-jB signaling. This miRNA may prove to be a new biomarker for assessing response to specific therapies for MM. Acknowledgments Grant support: the National Natural Science Foundation of China (81301498; 81271920); Jiangsu Provincial Program for Medical Innovation Teams and Leading Talents (LJ201133); Scientific Research Subject of Jiangsu Province Health Department (H201422), and the six major human resources project of Jiangsu Province (20012-WS-119), Translational Medicine Project of Affiliated Hospital of Nantong University (TDF-zh201407). Conflict of interest
The authors have no conflicts of interests.
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