Upregulation of microRNA-181b inhibits CCL18-induced breast cancer cell metastasis and invasion via the NF-κB signaling pathway

Abstract

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.

Keywords: apoptosis; breast cancer cell; chemokine ligand 18; microRNAs; migration.

Figure 1.

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.

Figure 2.

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.

Figure 3.

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.

Figure 4.

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.