OL Oncology Letters 1792-1074 1792-1082 D.A. Spandidos 10.3892/ol.2016.5230 OL-0-0-5230 Articles Upregulation of microRNA-181b inhibits CCL18-induced breast cancer cell metastasis and invasion via the NF-κB signaling pathway WangLei 1 WangYu-Xia 2 ChenLi-Ping 1 JiMing-Li 3 Department of General Surgery, The First Affiliated Hospital of Xinxiang Medical University, Xinxiang, Henan 453100, P.R. China Department of Pathophysiology, Xinxiang Medical University, Xinxiang, Henan 453003, P.R. China Department of Physiology, Xinxiang Medical University, Xinxiang, Henan 453003, P.R. China Correspondence to: Professor Ming-Li Ji, Department of Physiology, Xinxiang Medical University, 601 East of Jinsui Road, Xinxiang, Henan 453003, P.R. China, E-mail: mli_ji@tom.com 12 2016 05 10 2016 12 6 4411 4418 28072015 01092016 Copyright: © Wang et al. 2016 This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

The purpose of the present study was to investigate the effects of upregulating microRNA (miR)-181b expression in tumor-associated macrophages regarding breast cancer cell metastasis and to identify the target gene. Ectopic miR-181b was transfected into MDA-MB-231 and MCF-7 breast cancer cell lines with or without chemokine ligand 18 (CCL18) stimulation. Cell proliferation, migration/invasion and apoptosis rate were investigated. The binding effects of miR-181b to the 3′-untranslated region (UTR) of the nuclear factor (NF)-κB gene were detected with the dual luciferase reporter system. Immunofluorescent staining of the NF-κB key component P65 was performed. The messenger (m) RNA and protein expression of NF-κB induced by CCL18 with or without miR-181b stimulation was evaluated with reverse transcription-quantitative polymerase chain reaction and western blot analysis. When compared with the CCL18-stimulated group, miR-181b mimic-transfected cells exhibited significantly inhibited proliferation and migration, with an increased cell apoptosis percentage in a dose-dependent manner. Furthermore, the luciferase activity was reduced for cells with NF-κB 3′-UTR wild-type that were co-transfected with miR-181b mimics. Immunofluorescent staining of NF-κB demonstrably weakened the P65 signal in stimulated miR-181b mimic cells when compared with parental and CCL18-treated cells. The increased expression level of NF-κB induced by CCL18 in MDA-MB-231 and MCF-7 cells was suppressed by miR-181b mimics. Overexpression of miR-181b suppressed cell survival rate and migration. This overexpression may achieve this goal by regulating the NF-κB pathway in breast cancer cells. Our study demonstrated a potential therapeutic application of miR-181b in the treatment of breast cancer.

 breast cancer cell microRNAs chemokine ligand 18 migration apoptosis Introduction

Although breast cancer is commonly considered to be one of the most treatable types of malignancies, metastasis that is characteristic of breast cancer remains attributable to the majority of mortalities (1,2). Tumor-associated macrophages (TAMs) are considered to be responsible for metastasis by secreting several inflammatory cytokines to affect the tumor microenvironment (3). TAMs can remodel the cellular matrix and ultimately promote invasion and metastasis in tumor cells without the requirement for the presence of innately malignant cells (4). M2 macrophages are the main type of TAMs in breast cancer, and are associated with adverse outcomes in patients (5). Besides the well-known production of factors such as epidermal growth factor, vascular endothelial growth factor, and matrix metalloproteinase (6,7), chemokine ligand 18 (CCL18), a chemokine secreted by TAMs, has been confirmed as a promoter of the invasive cell phenotype and metastasis of breast cancer cells (8). However, the questions of how to regulate the adverse effects of CCL18 on cells as well as how to achieve optimal clinical outcomes remain unanswered.

In solid tumors, particularly in breast cancer, alterations in microRNA (miRNA or miR) expression have been recently shown to contribute to the pathogenesis of cancer, including metastasis (9). miRNAs are small, endogenous, non-coding, single-stranded RNA molecules containing 22 nucleotides, which can bind to a multitude of genes that are involved in the control of various cellular biological processes such as cell survival, cell migration/invasion and cell response to pro-inflammatory factors (10,11). miRNAs can achieve these regulatory roles by affecting their target's gene expression at the post-transcriptional level in a tissue and in a developmental stage-specific manner (12–14). Previous studies have suggested that miR-181b can be used as a diagnostic and prognostic biomarker in breast cancer (15–17). Despite the different expression levels reported from multiple studies, miR-181b has been observed to exhibit regulatory effects during breast cancer progression (18–20). In addition, previous studies have indicated that miR-181b may inhibit breast cancer cell metastasis by targeting the inflammatory cytokines chemokine (C-X-C motif) ligand (CXCL)-1 and CXCL-2 (21).

The present study aims to explore the function of miRNA-181b mimics in the TAM cytokine CCL18, and to evaluate the role that it plays in inducing breast cancer cell metastasis. The current study also explores the biological character of these cells and the relevant mechanisms involved.

 Materials and methods <sec> <title>Cell lines

The human breast cancer MDA-MB-231 and MCF-7 cell lines (American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany), 0.05% L-glutamine, 100 UI/ml penicillin and 50 µg/ml streptomycin (Merck Millipore) in a humidified atmosphere with 5% CO2 at 37°C. Regular split process was conducted, and cells on 20 passages were selected for subsequent experiments. All tested cells were supplied with 1% FBS and then treated with 50 ng/ml human CCL18 (Immuno Tools GmbH, Friesoythe, Germany) overnight for designed experiments. All cell lines had experienced short tandem repeats authentication test and no report of mycoplasma contamination.

 Cell transfection

For transient transfection, cells were plated at a density of 1×106 cells/ml and transfected with miR-181b and a negative control (Genepharm, Inc., Sunnyvale, CA, USA) using a Lipofectamine™ 2000 transfection reagent (Ambion; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. Transfection efficiency was validated by quantitative polymerase chain reaction (qPCR) as described below.

 Cell viability assay

The methylthiazolyldiphenyl-tetrazolium bromide (MTT) method was performed to test cell viability. In total, 1×104 cells were seeded in 96-well plates for 48 h, and 100 µl MTT (Sigma-Aldrich; Merck Millipore) was added to each well. The plates were incubated at room temperature until purple formazan crystals had formed. Each well was washed with 100 µl dimethyl sulfoxide and was air dried at room temperature for 30 min. The cell viability was quantified at the absorbance values of 490 nm using a universal microplate spectrophotometer (Thermo Fisher Scientific, Inc.).

 qPCR

RNA samples were extracted from the MDA-MB-231 and MCF-7 cell lines with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and stored at −80°C. Reverse transcription (RT)-qPCR was performed using the PrimeScript RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China). The RT reaction mixture (total volume, 10 µl) included 2 µl 5X PrimeScript™ buffer (Clontech Laboratories, Inc., Mountainview, CA, USA), 0.5 µl PrimeScript™ RT Enzyme Mix I (Clontech Laboratories, Inc.), 0.5 µl oligo (dT) primer (50 µM), 0.5 µl random 6-mers (100 µM) and 6.5 µl RNase-free distilled H2O. qPCR was completed using the 7900HT Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) with SYBR green fluorescent dye and the following cycling conditions: 95°C for 10 sec, 59°C for 20 sec and 72°C for 10 sec, for 40 cycles. U6 RNA expression was selected as the internal control for miR-181b, while glyceraldehyde 3-phosphate dehydrogenase was selected as the internal control for NF-κB.

The primers for NF-κB were as follows: Forward, 5′-ACCAACGCTTATGCATTTAAATT-3′ and reverse, 5′-CTCGATCAGGAGCCACTCATTAAT-3′. The primers for miR-181b were as follows: Forward, 5′-CCCAAGCTTTGATTGTACCCTATGGCT-3′ and reverse, 5′-CGGGGTACCTGTACGTTTGATGGACAA-3′. The relative expression was calculated according to the 2−ΔΔCq method, where Cq is the quantification cycle to detect fluorescence (22). The experiments were repeated ≥3 times.

 Migration and invasion assay

Trypsinized cells in different groups were first collected from culture flasks. In total, 200 µl of cell suspension at a density of 1×106 cells/ml was added to the upper chamber of Transwell plates (Corning Life Sciences, Lowell, CA, USA) in six wells. During the invasion test, the Transwell was coated with 100 µl Matrigel. Subsequently, 600 µl RPMI 1640 culture medium was added to the lower chamber supplied with 10% FBS, and incubated for 24 h for the migration test (or for 48 h for invasion detection) at 37°C. Migrated and invasive cells were stained with crystal violet. In total, five randomly selected views under a microscope were counted.

 Cell apoptosis

The apoptosis percentage of cells in response to different treatments was detected using a staning kit containing annexin V conjugated to fluorescein isothiocyanate (FITC) and propidium iodide (BD Pharmingen, San Diego, CA, USA). Hoechst 33342 (Sigma-Aldrich; Merck Millipore) staining was additionally performed and evaluated using a fluorescence microscope (Nikon Corporation, Tokyo, Japan) with an excitation wavelength of 350 nm and an emission wavelength of 460 nm to validate and visualize nuclear apoptosis features.

 Immunofluorescence examination of nuclear factor (NF)-κB

In total, 1×105 cells were collected and placed in cold phosphate-buffered saline (PBS) for 5 min supplied with 2.5 mg/ml Triton X-100, and then washed with PBS three times. Cells were next incubated with a primary anti-NF-κB antibody (1:500; Cat No. 622602; BioLegend Inc., San Diego, CA, USA) with 100 ml FBS at 4°C overnight prior to incubation with a secondary immunoglobulin G antibody conjugated to FITC (1:10,000; Cat No. F9512; Sigma-Aldrich; Merck Millipore) for 1 h at room temperature. Immunofluorescent staining of NF-κB was examined under a confocal laser scanning microscope (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

 Luciferase reporter assay to validate the miR-181b target gene

The present study first performed a potential target gene search using multiple target gene prediction softwares (microRNA, www.microrna.org; TargetScan, www.targetscan.org; and miRDB, www.mirdb.org). The identified candidate target gene by all three softwares (NF-κB) was then analyzed in a molecule annotation system to forecast biological function. To validate the direct binding effect of miR-181b on its target gene NF-κB, the present study performed a luciferase reporter assay using a commercial dual-luciferase reporter test kit (Promega Corporation, Madison, WI, USA) on wild-type NF-κB and mutant NF-κB. In addition, CCL18 was supplemented into the culture medium to observe alterations in NF-κB levels. The sequences used were as follows: miR-181b mature sequence, 3′-UGGGUAGCAGUCGUUACUUACAA-5′; and putative miR-181b-binding site sequence in the 3′-UTR of NF-κB wild-type, 5′-GUUGUCGUCAGUCAGCAAUGACUUGGGACUAC-3′ and NF-κB mutant, 5′-GUUGUCCGUAGUCAGCAAUCUGUUGGGACUAC-3′.

 Western blot analysis

All procedures were conducted according to the manufacturer's protocol of the antibodies listed below (Abcam, Cambridge, UK). Cells were centrifuged at 1,000 × g at 4°C for 10 min and lysed in a lysis solution (Cat No. ab152163; Abcam). The proteins were loaded into the wells of an 8% sodium dodecyl sulfate-polyacrylamide electrophoresis gel, along with molecular weight markers (Cat No. ab116028; Abcam). The proteins were then electro-transferred onto nitrocellulose membranes. The membrane was blocked for 1 h at room temperature, or alternatively overnight at 4°C, with 5% blocking solution (Cat No. ab126587; Abcam). The membranes were incubated with primary antibodies against β-actin (Cat No. ab8227; Abcam), NF-κB P50 (Cat No. ab32360; Abcam) and NF-κB P65 (Cat No. ab16502; Abcam) at 1:1,000 dilution overnight at 4°C, and horseradish peroxidase-conjugated secondary antibody (1:10,000; Cat No. ab97023; Abcam) was then added for 1 h at 37°C. β-actin was used as an internal control, based on the relative intensities of the bands. The proteins were visualized using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Inc.) and were analyzed quantitatively by ImageJ Software version 1.48 (National Institutes of Health, Bethesda, MD, USA).

 Statistics

Comparisons of different experimental groups were performed using SPSS software version 19.0 (IBM SPSS, Armonk, NY, USA) and GraphPad Prism 5.01 (GraphPad Software, Inc., La Jolla, CA, USA). The two-tailed Student's t-test was used for comparisons. Experiments were repeated ≥3 times, and data were presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

 Results <sec> <title>miR-181b inhibits cell proliferation induced by CCL18 in cell lines

As shown in CCL18 induced an increase in MDA-MB-231 cell proliferation (P=0.021 at 36 h, P=0.014 at 48 h and P=0.007 at 72 h; Fig. 1A) when compared with the control, while miR-181b mimics at 50 µg/ml (P=0.032 at 36 h, P=0.024 at 48 h and P=0.011 at 72 h; Fig. 1A) and 100 µg/ml (P=0.022 at 36 h, P=0.017 at 48 h and P=0.006 at 72 h; Fig. 1A) significantly inhibited cell proliferation in a dose-dependent manner when compared with CCL18-stimulated cells. miR-181b transfection efficiency in MDA-MB-231 cells was validated by RT-qPCR (P=0.003, compared with the control; Fig. 1B). The breast cell line MCF-7 exhibited similar effects, which included significantly increased cell proliferation following CCL18 treatment (P=0.013 at 36 h, P=0.020 at 48 h and P=0.008 at 72 h; Fig. 1C) when compared with the control, and decreased cell proliferation following miR-181b mimics treatment at 50 µg/ml (P=0.041 at 36 h, P=0.027 at 48 h and P=0.016 at 72 h; Fig. 1C) and 100 µg/ml (P=0.019 P=0.015 at 36 h, P=0.005 at 48 h and P=0.006 at 72 h; Fig. 1C) compared with CCL18-stimulated cells. miR-181b transfection efficiency in MCF-7 cells was validated by RT-qPCR (P=0.011; Fig. 1D).

 miR-181b mimic inhibits cell migration and invasion induced by CCL18

The cells invading through the filter were stained with crystal violet (Fig. 2A and B). In the miR-181b mimic group, the number of cells crossing the filter was lower compared with that in the CCL18-stimulated group (151. 2±21.3 vs. 93.8±10.1 for MDA-MB-231 cells, P=0.009; and 75.4±18.2 vs. 20.0±6.7 for MCF-7 cells, P=0.033; Fig. 2C). The relative cell invasion number in the miR-181b mimic group was reduced (142.6±20.1 vs. 81.4±9.0 for MDA-MB-231 cells, P=0.012; and 66.2±14.4 vs. 17.8±6.8 for MCF-7 cells, P=0.014; Fig. 2D). These results indicate that the migration and invasion capabilities of the cells in the miR-181b mimic group were lower compared with those of the cells in the CCL18-stimulated group.

 miR-181b induces cell apoptosis in CCL18-stimulated cells

In comparison with the control, CCL18 stimulation had no effect on cell apoptosis. Following miR-181b treatment, the early and late apoptosis rates increased among all the experimental cell lines (P=0.025 for MDA-MB-231 cells and P=0.018 for MCF-7 cells; Fig. 3A-E). Additionally, Hoechst 33342-stained cells demonstrated visual apoptosis morphology changes (Fig. 3F). These results confirmed that the rate of apoptosis had increased in miR-181b mimic-transfected cells compared with control cells.

 CCL18 inhibits miR-181b expression through the NF-κB signaling pathway

The present results demonstrated that the signal intensity of P65 (one of the NF-κB transcriptional expression representatives) (23) was significantly reduced by the miR-181b mimic, but was increased by stimulation with CCL18, when compared with the controls in the two cell lines (P=0.022 for MDA-MB-231 cells and P=0.019 for MCF-7 cells; Fig. 4A). In addition, the mRNA levels of NF-κB increased upon CCL18 stimulation in MDA-MB-231 and MCF-7 cells, while miR-181b expression decreased the NF-κB expression levels (P=0.009 for MDA-MB-231 cells and P=0.007 for MCF-7 cells; Fig. 4B). The luciferase reporter results validated the direct regulation effect of miR-181b on NF-κB (Fig. 4C). Furthermore, the protein expression levels of the NF-κB components P65 and P50 decreased subsequent to transfection with miR-181b mimics compared with CCL18 treatment (P=0.034 for P65 and P=0.027 for P50; Fig. 4D).

 Discussion

Altered miR-181b expression has been detected in numerous types of cancer, but the expression levels differ (24). Previous studies have reported the downregulation of miR-181b in lung cancer, while others have revealed upregulation in acute myeloid leukemia (25,26). These studies indicate that the expression of miR-181b may be cell specific. Despite the inconsistent expression pattern, it is commonly accepted that miR-181b regulates cell inflammatory cytokines and additionally affects cancer processes and clinical outcomes (27,28). The present results demonstrate that miR-181b has the ability to reverse CCL18-induced breast cancer cell metastasis in vitro. Upregulation of miR-181b significantly suppressed cell proliferation, migration and invasion. Additional target identification indicated that miR-181b achieved these effects on breast cancer cells through regulating the NF-κB signaling pathway.

A previous study conducted in ductal breast adenocarcinoma patient samples demonstrated that there is a positive correlation between CCL18 expression in TAMs from breast tissue and tumor invasiveness, when compared with healthy control donor samples (8). The present study revealed that CCL18 stimulation markedly promoted cell proliferation, migration and invasion, when compared with the untreated controls, in two commonly used breast cancer cell lines. These results suggest that CCL18 plays a role in promoting the invasive cell phenotype and enhances metastasis.

Previous studies have reported the link between NF-κB and malignant cell mobility (29–31). The production of CCL18 by TAMs induces NF-κB activation (32). By contrast, activated NF-κB has the ability to upregulate inflammatory cytokines such as CCL18 and to disturb the microenvironment of cancer cells (33). In addition, NF-κB is also regulated by miRNAs such as miR-181b (34–36). There has been no direct evidence of an association between miR-181b and CCL18; however, it is known that miRNAs are important in regulating macrophages in various types of cancer (37). Considering the innate link between NF-κB and miR-181b, the present study additionally examined the possible effects of miR-181b on CCL18-induced metastasis. Firstly, the results revealed that miR-181b reversed the cell invasiveness induced by CCL18. Secondly, evidence was identified to support the direct binding effect between miR-181b and NF-κB. Taken together, these results suggest that miR-181b plays a crucial role in CCL18-induced metastasis by suppressing NF-κB expression. A previous study reported that miR-181b participates in the cell response to pro-inflammatory stimuli by affecting downstream NF-κB signaling (38). In that study, the restoration of miR-181b inhibited NF-κB signaling in the vascular endothelium, and decreased lung injury and mortality in endotoxemic mice (38). Another study reported that miR-181a (a similar molecule to miR-181b, which belongs to the same miR-181 family) significantly inhibited the increase in inflammatory factors, including interleukin (IL)-1b, IL-6 and tumor necrosis factor-a in cells (39). It is well known that IL-6 and CCL18 have a close association in the response to cell migration (33). IL-6 and CCL18 have been reported to be significantly decreased in breast cancer samples (39,40). One study has also identified an inverse correlation between macrophages secretion and miR-181b expression (41). The authors also provided evidence that upregulation of miR-181b was associated with NF-κB (41).

In the present study, it was also observed that the cell apoptosis percentages increased in the miR-181b mimic-stimulated group. This result may be explained by the regulatory role of NF-κB in apoptosis (23). It is well documented that, during tumor progression, a large release of inflammatory cytokines such as CCL18 and IL-6 in the microenvironment of a solid tumor enhances NF-κB activity and induces the expression of anti-apoptotic genes, which in turn promotes tumor cell proliferation, invasion and metastasis (42,43). miR-181b inhibited NF-κB expression, and thus accelerated cancer cell apoptosis (44). Notably, the present study provided preliminary evidence that specific miRNAs may affect TAMs activity. To validate this direct effect, additional miRNAs and TAM-derived cytokines should be evaluated in future studies. It has been noted that the expression of miRNAs in cancer cells may disturb the production of cytokines in the tumor immune environment, thus influencing the activation of signal transduction pathways (45,46). The association of the expression levels of these cytokines and miRNA overexpression/knockdown models may provide more direct information on the regulatory role of miRNAs in cytokines production.

In conclusion, the present results provide evidence that miR-181b is a cytokine-responsive miRNA that serves a role in regulating the expression of key NF-κB genes that are involved in the response to inflammatory cytokines in breast cancer. Overexpression of miR-181b may provide a potential therapeutic approach to treat breast cancer metastasis.

 Acknowledgements

The present study was funded by the Program of Natural Science Foundation of Henan Education Committee (Zhengzhou, China; grant no. 13A320854).

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Alteration of cell proliferation in a time- and dose-dependent manner in two cell lines. (A) MDA-MB-231 cells exhibited significantly decreased cell growth rates in the group treated with miR-181b (100 µg/ml) for 72 h. By contrast, CCL18 (50 ng/ml)-stimulated cells started to grow at 48 h. (B) miR-181b transfection efficiency was validated by reverse transcription-quantitative polymerase chain reaction in the MDA-MB-321 cell line. (C) The MCF-7 cell line demonstrated the same growth pattern as the MDA-MB-231 cell line. (D) miR-181b transfection efficiency was validated in MCF-7 cells. *P<0.05 compared with the control group. All experiments were conducted in triplicate. CCL18, chemokine ligand 18; miR, microRNA; OD, optical density; NC, negative control.

Migration and invasion assay following differential treatment. (A) Cell migration assay with crystal violet staining revealed a decreased migration ability in the miR-181b mimic group compared with that observed in the group exposed to CCL18 stimulation (50 ng/ml) (magnification, ×200). (B) Cell invasion assay with crystal violet staining revealed a decreased migration ability in the miR-181b mimic group compared with that observed in the group exposed to CCL18 stimulation (50 ng/ml) (magnification, ×200). (C) Quantification of the results for migration and invasion cells counts for the MDA-MB-231 cell line. (D) Quantification of the results for migration and invasion cells counts for the MCF-7 cell line. *P<0.05 compared with control cells. CCL18, chemokine ligand 18; miR, microRNA.

Apoptosis distribution in two breast cell lines following treatment. (A-D) Flow cytometry images for differentially treated cells, where (A) represents control, (B) represents MDA-MB-231 cells treated with miR-181b, (C) represents CCL18 stimulation (50 ng/ml) and (D) represents MCF-7 cells treated with miR-181b mimic. (E) Quantification of the distribution of apoptosis in the two cell lines. (F) Representative images of condensation or apoptotic bodies, reflecting the typical apoptotic nuclear morphology (arrow), upon transfection with miR-181b mimics and Hoechst 33342 staining (magnification, ×200). *P<0.05 compared with the control cells. CCL18, chemokine ligand 18; miR, microRNA; FITC, fluorescein isothiocyanate.

Target verification of miR-181b. (A) Immunofluorescence images of the expression of the NF-κB marker P65 following differential treatment. Cells were stained with an anti-P65 antibody (green). Co-staining with 4′,6-diamidino-2-phenylindole was used to visualize the nuclei (blue). Merged images of the two types of staining are also shown (magnification, ×400). (B) miR-181b and NF-κB messenger RNA expression levels subsequent to CCL18 stimulation. (C) Luciferase activities of differentially treated cells. (D) Expression of NF-κB-associated proteins subsequent to CCL18 and miR-181b mimic stimulation. *P<0.05 compared with the control cells. CCL18, chemokine ligand 18; miR, microRNA; NF-κB, nuclear factor-κB; Mut, mutant.