miR-206 Promotes Cancer Progression by Targeting Full-Length Neurokinin-1 Receptor in Breast Cancer

Abstract

Substance P plays a pivotal role in human cancer development and progression by binding to its receptor, neurokinin-1. Neurokinin-1 has 2 isoforms: full-length neurokinin-1 and truncated neurokinin-1, the latter lacking the cytoplasmic terminal 96-amino acid residues of the full-length protein. We have identified 3 candidate miR-206 target sites within the 3'-untranslated region of the full-length neurokinin-1 gene from bioinformatics database searches. In the present study, real-time quantitative polymerase chain reaction was performed to quantify the expression of miR-206, and the expression of neurokinin-1 and full-length neurokinin-1 was detected by immunohistochemistry in 82 clinical cases of breast cancer and paired adjacent normal tissues. The miR-206 target gene was demonstrated by using a dual-luciferase reporter assay, quantitative real-time polymerase chain reaction, and Western blotting. Transwell migration and invasion, colony formation, and proliferation assays were performed to evaluate the effects of miR-206 expression on various aspects of breast cancer cell behavior in vitro. We showed that miR-206 expression is upregulated in breast cancer cell lines and breast cancer tissues when compared to that in adjacent normal tissues, and full-length neurokinin-1 expression inversely correlates with Tumor Lymph Node Metastasis (TNM) stage and lymph node metastasis. Western blotting, quantitative real-time polymerase chain reaction, and dual-luciferase reporter assays demonstrated that miR-206 binds the 3'-untranslated region of full-length neurokinin-1 messenger RNA, regulating protein expression. We showed that the overexpression of miR-206 promotes breast cancer cell invasion, migration, proliferation, and colony formation in vitro. The present study furthers the current understanding of the mechanisms underlying breast cancer pathogenesis and may be useful for the development of novel targeted therapies.

Keywords: breast cancer; invasion; microrna-206; neurokinin-1 receptor; proliferation.

Figure 1.

Immunohistochemical analysis of total NK1R and NK1R-FL protein expression in breast tumor tissues (T) and paired adjacent normal tissues (ANT). (A and B) Positive staining of total NK1R in ANT and breast tumor tissues (at ×100). (C) Positive staining of NK1R-FL in ANT (at ×100). (D) Weak staining of NK1R-FL in breast tumor tissues (at ×100). The expression levels of NK1R-FL and miR-206 in human breast tissues and cell lines. (E and F) Average expression level of NK1R-FL and miR-206 in human breast tumor tissues (n = 82) and adjacent normal tissues (n = 82). mRNA and miR-206 abundance was assessed by real-time quantitative PCR and normalized to β-actin or U6 RNA. (G and H) Real-time quantitative PCR analysis of NK1R-FL and miR-206 in human breast cancer cell lines (metastatic breast cancer cell lines MDA-MB-231, T47D, MCF-7, and SK-BR-3) and nontumorigenic epithelial cell line MCF-10A. *P < .05 versus normal control. (I) The expression levels of NK1R-FL in human breast cancer cells (metastatic breast cancer cells MDA-MB-231, T47D, MCF-7, and SK-BR-3) and MCF-10A in nontumorigenic epithelial cells were detected by Western blotting.

Figure 2.

miR-206 downregulation NK1R-FL expression. (A) A1, A2, and A3 Discovery of 3 potential miR-206 target sites within the 3′-UTR of NK1R-FL according to the bioinformatics analysis. A putative miR-206 binding target sites in the 3′-UTR of NK1R-FL mRNA among mammalian species (upper panel); site-directed mutations in the sequence complimentary to the seed region for miR-206 (lower panel). (B and C) Relative luciferase activity of NK1R-FL-3′-UTR-WT and NK1R-FL-3′-UTR-MUT in SK-BR-3 and MCF-10A cells cotransfected with miR-206 mimic or negative control, and in MDA-MB-231 cells with As-miR-206 or inhibitor control. (D, E, and F) Protein levels of NK1R-FL were determined by western blotting in (D, E) SK-BR-3 and MCF-10A cells transfected with miR-206 mimic or negative control; MDA-MB-231 cells transfected with As-miR-206 or inhibitor control (F). (G and H) After transfection, real-time quantitative PCR was used to examine the mRNA expression of NK1R-FL in SK-BR-3, MCF-10A and MDA-MB-231 cells. *P < .05, **P < .01, ***P < .0005, ****P < .0001 versus normal control.

Figure 3.

Effect of activated NK1R-FL on intracellular Ins(1,4,5)P3 response and ERK1/2 phosphorylation. (A, B, and C) The rise of intracellular IP3 in cells that express NK1R-FL or NK1R-Tr. Cells transfected with miR-206 or As-miR-206 were stimulated with 10⁻⁷ mol/L SP only or treated for 30 minutes with the NK1R antagonist Aprepitant after that. (D, F, and H) SK-BR-3, MCF-10A, and MDA-MB-231 cells that express NK1R-FL or NK1R-Tr transfected with miR-206 or As-miR-206, were treated for 30 minutes with the NK1R antagonist Aprepitant and then stimulated by 10⁻⁷ mol/L SP or untreated as control. (E, G, and I) SK-BR-3 and MCF-10A cells that express NK1R-FL or NK1R-Tr were transfected with miR-206 mimic or negative control, and in MDA-MB-231 cells, with As-miR-206 or inhibitor control or untransfected as a control. Phospho-ERK1/2 was detected by antiphospho-ERK1/2 antibody.

Figure 4.

miR-206 promoted human breast cancer cell migration, invasion, proliferation, and colony formation. Growth of the SK-BR-3 cells transfected with miR-206 mimic or negative control (NC), and in MDA-MB-231 cells, with As-miR-206 or inhibitor control at an indicated time (at ×100). (A and B) Transwell assays on SK-BR-3 and MDA-MB-231 cells were employed to analyze the effect of miR-206 on migration. For the invasion assay, 1:3 Matrigel was added. **P < .01, ***P < .0005, ****P < .0001. (C) Representative photomicrographs of colony formation assay of SK-BR-3 and MDA-MB-231 cells with the transfected miR-206 ***P < .0005, ****P < .0001. (D and E) The cell counting kit-8 assay was performed to analyze the effect of miR-206 on proliferation in SK-BR-3 and MDA-MB-231 cells. Data are presented as the mean ± SD from 3 independent experiments. **P < .01 versus normal, control ****P < .0001 versus normal. The agomirs of miR-206 and their controls were inoculated into nude mice carrying MDA-MB-231 xenografts (n = 5/group). As shown in Figure 4F, tumors transfected with miR-206 agomirs grew significantly faster than the control tumors at different time points (*P < .05), which was consistent with the present in vitro cell proliferation results. Four weeks later, the weights of tumors in mice transfected by miR-206 silencing cells were significantly increased compared to control cells (**P < .01, Figure 4G).