MiR‐34b/c‐5p and the neurokinin‐1 receptor regulate breast cancer cell proliferation and apoptosis

Abstract Objectives MiR‐34 is a tumour suppressor in breast cancer. Neurokinin‐1 receptor (NK1R), which is the predicted target of the miR‐34 family, is overexpressed in many cancers. This study investigated the correlation and clinical significance of miR‐34 and NK1R in breast cancer. Materials and Methods Western blotting, quantitative reverse transcription‐PCR (qRT‐PCR) and luciferase assays were conducted to analyse the regulation of NK1R by miR‐34 in MDA‐MB‐231, MCF‐7, T47D, SK‐BR‐3 and HEK‐293 T cells. MiR‐34b/c‐5p, full‐length NK1R (NK1R‐FL) and truncated NK1R (NK1R‐Tr) expression in fifty patients were quantified by qRT‐PCR and correlated with their clinicopathological parameters. CCK‐8 assays, colony formation assays and flow cytometry were used to measure cell proliferation and apoptosis in MDA‐MB‐231 and MCF‐7 cells transfected with miR‐34b/c‐5p or NK1R‐siRNA and before treatment with or without Substance P (SP), an endogenous peptide agonists of NK1R. The effect of NK1R antagonist aprepitant was also investigated. In vivo xenograft models were used to further verify the regulation of NK1R by miR‐34b/c‐5p. Results Expression levels of miR‐34b/c‐5p and NK1R‐Tr, but not NK1R‐FL, were associated with enhanced malignant potential, such as tumour stage and Ki67 expression. The overexpression of miR‐34b/c‐5p or NK1R silencing potently suppressed cell proliferation and induced G2/M phase arrest and the apoptosis of MDA‐MB‐231 and MCF‐7 cells. The NK1R antagonist aprepitant had similar effects. In vivo studies confirmed that miR‐34b/c‐5p overexpression or NK1R silencing reduced the tumorigenicity of breast cancer. In addition, SP rescued the effects of miR‐34b/c‐5p overexpression or NK1R silencing on cell proliferation and apoptosis in vitro and in vivo assays. Conclusions MiR‐34b/c‐5p and NK1R contribute to breast cancer cell proliferation and apoptosis and are potential targets for breast cancer therapeutics.

MicroRNAs (miRNAs) are a class of endogenous, small, noncoding RNAs. They modulate cell behaviours, proliferation, differentiation and apoptosis by simultaneously targeting multiple genes. [2][3][4][5] In breast cancer, the miR-34 family is often lost or poorly expressed and is a potential tumour suppressor gene. 4,6 MiR-34 may inhibit breast cancer cell proliferation by suppressing c-Myc and the phosphatase activities of ERK and AKT. 7,8 Moreover, miR-34 induces cell cycle arrest and apoptosis in breast cancer. 4,9 These findings strongly suggest that miR-34, as a tumour suppressor, may be a new therapeutic target and intervention strategy against breast cancer.
G protein-coupled receptors (GPCR) are overexpressed in various cancers and contribute to cell growth when they are activated by circulating or locally produced ligands; they are also used as a tumour diagnosis signal, [10][11][12] although the underlying molecular mechanisms remain poorly understood. One such GPCR is neurokinin-1 receptor (NK1R) 13,14 and its specific endogenous peptide agonist is neuropeptide Substance P (SP). 15 Evidence indicates that the biological action of SP is primarily mediated by its binding to NK1R. 16 The SP-NK1R signalling pathway is linked to the pathophysiological processes of pain transmission, 7 chemotherapy-induced vomiting 17 and inflammation. 18 There is a close correlation between SP-NK1R and tumour development. 15,19,20 NK1R expression is elevated in glioblastoma 15 and pancreatic, 21 breast 22,23 and gastric cancers. 24 SP promotes cell proliferation and increases collagen I expression. 25 NK1R agonists promote glioblastoma cell proliferation and migration, whereas the NK1R antagonists L732138 and SPA induce cell apoptosis. 15 Moreover, another NK1R antagonist, aprepitant, induces robust inhibition of tumour growth and apoptosis in a wide variety of cancers. [26][27][28][29] NK1R has two structural isoforms, the fulllength receptor (NK1R-FL) and the truncated receptor (NK1R-Tr). In addition to their structural differences, the two isoforms also exert different biological functions. We and others have demonstrated that NK1R-Tr expression is upregulated in cancer. 22,23 Our previous study shows that NK1R-Tr is strongly overexpressed in breast cancer. Furthermore, NK1R is a putative target of miR-34, as determined by analyses using several bioinformatics databases such as TargetScan, miRbase and RNAhybrid. The aim of our study was to investigate the biological roles of miR-34 and NK1R in breast cancer. We also explored the potential regulation of NK1R by miR-34 and their potential as therapeutic targets for breast cancer patients.
In this study, we found that the expression of NK1R-Tr was markedly upregulated and that the levels of miR-34b/c-5p and NK1R-FL were downregulated in human breast cancer cell lines and tumour tissues. However, miR-34b/c-5p and NK1R-Tr, but not NK1R-FL, were significantly associated with the clinical features of breast cancer. However, the inhibition of cell proliferation by miR-34b/ c-5p overexpression was primarily consistent with the silencing of NK1R-Tr, but not NK1R-FL, in breast cancer and miR-34b/c-5p overexpression and NK1R-Tr silencing both induced cell cycle G2/M arrest and apoptosis; furthermore, SP only partly rescued this effect.
Aprepitant, an antagonist of NK1R, also inhibits cell proliferation and induces cell cycle G2/M phase arrest and apoptosis. Finally, animal experiments further confirmed the tumour inhibition effects of miR-34b/c-5p and NK1R silencing. Together, our data indicate that miR-34b-5p, miR-34c-5p and NK1R-Tr play important roles in the initiation and proliferation of human breast cancer, and they may be potential targets for human breast cancer treatments.

| Cell transfection and luciferase reporter assay
The specific siRNA sequences of NK1R and negative controls were purchased from Invitrogen and miR-34a-3p, miR-34a-5p, miR-34b-3p, miR-34b-5p, miR-34c-3p, miR-34c-5p mimics, inhibitors, mimic control and inhibitor control were purchased from RiboBio. Cells were cultured in six-well plates until they were in the logarithmic growth phase and then transfected with either 100 pmol/well siNK1R and miRNA mimics or 400 pmol/well miRNA inhibitors using reporters for NK1R-FL were also tested. To investigate whether SP induced NK1R signalling, SP treatment was coupled to luciferase activity, which was measured. Briefly, cells were transfected with the reporter plasmid PNK1R-luc. Twenty-four hour later, the transfected cells were seeded in a 96-well plate at a density of 30 000 and cultured for another 24 hour. The cells were exposed to 100 nmol/L SP for 8 h at 37°C. Untreated cells were used as a control. Luciferase activities were measured using a luciferase assay system.

| Western blotting
After SP treatment for 48 h, cells were rapidly washed with chilled and then blots were developed using ECL (Millipore). β-Actin was used as internal control.

| CCK-8 assay
Cell suspensions were seeded in 96-well plates at an initial density of Kumamoto, Japan) for 2 hours in a humidified atmosphere (37°C, 5% CO 2 ) in the dark, followed by replacement with fresh culture medium. The absorbance of each sample was measured at 450 nm using a universal microplate reader (Bio-Tek). All experiments were performed in triplicate.

| Colony formation
Cells were plated in 35-mm plates (5 × 10 2 cells per plate) after treatment with SP or aprepitant and were cultured in a humidified

| Cell cycle analysis
Cells were treated with or without SP or chemical inhibitors as indicated and then processed for cell cycle analysis using flow cytometry. Briefly, cells were fixed in 75% ethanol at 4°C overnight. After

| Immunohistochemical staining
Immunohistochemistry was performed on paraformaldehyde-fixed paraffin sections. The primary antibody of Ki67 (1:100) was purchased from Beijing Zhongshan Biotechnology Inc (Beijing, China).

| Statistical analysis
All results were performed using SPSS software for Windows version 23.0 (IBM, SPSS, Chicago, IL, USA). Statistically significant differences between groups were determined using the Student's two-tailed t test or the two-way ANOVA test. The association between pairs of variables was determined using Spearman order correlations. The IC 50 of aprepitant was calculated using the regression straight line function based on the least squares technique. All experiments for cell culture were performed at least in triplicate. The results were expressed as the mean ± SD. In all cases, P < 0.05 was considered statistically significant.

| MiR-34b/c-5p and NK1R-Tr expression is associated with the clinicopathological parameters of breast cancer
To investigate the relationship between the expression of miR-34b/c-5p and NK1R and the clinicopathological parameters of breast cancer, the expression levels of miR-34b/c-5p, NK1R-FL and NK1R-Tr were examined by qRT-PCR in 50 paired samples of breast cancer and matched adjacent nontumour tissues. Significant differences in the expression of miR-34b/c-5p and NK1R were observed between tumour and adjacent nontumour tissues (Figure 2A,B). Among them, miR-34b/c-5p and NK1R-FL were significantly overexpressed in normal breast tissues (P < 0.001). 35 Conversely, NK1R-Tr expression was significantly overexpressed in breast cancer tissues (P = 0.0023). 22 As shown in Table 1, F I G U R E 5 MiR-34b/c-5p and NK1R regulate apoptosis in breast cancer MDA-MB-231 and MCF-7 cells. A, Apoptosis was evaluated after culturing MDA-MB-231 and MCF-7 cells transfected with miR-34b/c-5p or NK1R-siRNA, before treatment with or without SP and staining with Annexin V at 48 h. The flow cytometry profile depicts Annexin V/FITC staining on the x-axis and PI staining on the y-axis (left), the number represents the percentage of total apoptotic cells in each condition (right). B, Caspase-3, caspase-8 and caspase-9 activities were detected in MDA-MB-231 (left) and MCF-7 (right) cells transfected with miR-34b/c-5p or NK1R-siRNA with or without SP treatment. The data represent the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001  levels. There was no significant correlation between miR-34b/c-5p and NK1R-Tr expression and age and lymph node status ( Table 1, Table S1).
Together, these observations support the hypothesis that NK1R-Tr is closely associated with miR-34b/c-5p.

| MiR-34b/c-5p and NK1R regulate the proliferation of breast cancer cells
We found that SP was able to induce NK1R signalling in MDA-

MB-231 and MCF-7 cells in comparison with control groups as in-
dicated by our luciferase assay (P < 0.001, Figure S3) ing miR-34b/c-p had significantly suppressed proliferation rates compared with the control cells, as analysed by the CCK-8 assay (P < 0.05). SP treatment significantly impaired this effect ( Figure 3A).
These results were further confirmed by a colony formation assay ( Figure 3B). In addition, we also investigated whether aprepitant had potential antitumour efficacy ( Figure S4A,B), and we determined the IC 50 growth inhibition concentration of aprepitant needed for tumour cell lines (Table S2). Based on our previous study, NK1R-FL and NK1R-Tr have opposing roles in cell proliferation, and our present results further support the notion that miR-34b/c-5p inhibits cell proliferation by regulating NK1R-Tr

| MiR-34b/c-5p and NK1R regulate the G2/M phase transition in breast cancer
We conducted flow cytometry analysis to further investigate the potential mechanism of miR-34b/c-5p-and NK1R-mediated proliferation. As shown in Figure 4A, the overexpression of miR-34b/c-5p or NK1R silencing in MDA-MB-231 and MCF-7 cells significantly increased the percentage of cells in the G2/M phase (P < 0.01). 37 Furthermore, compared with the control group, after SP treatment, the percentage of cells in the G2/M phase decreased. In addition, high doses of aprepitant had more intense effects on cell cycle arrest (P < 0.001, Figure S5A). NK1R regulates cell proliferation by mediating the activity of the ERK1/2 and PI3K/AKT signalling pathways.
We examined the effect of miR-34b/c-5p and NK1R on the regulation of the phosphorylation activities of ERK and AKT. As shown in Figure 4B, the levels of p-ERK and p-AKT were decreased in miR-34b/c-5p-overexpressing or NK1R-silenced cells, but SP lessened their effects. Aprepitant also decreased the phosphorylation activity of ERK and AKT ( Figure S5B). We also investigated the function of miR-34b/c-5p and NK1R on cell cycle-related proteins. Cyclin B1 and CDC23 mRNA and protein expression were significantly reduced in NK1R-silenced and aprepitant-treated cells compared with control cells (P < 0.05; Figures S5B,C and S6). However, only CDC23, not cyclin B1, was decreased in miR-34b/c-5p overexpressing cells ( Figure 4B). In addition, cyclin B1 and CDC23 levels increased after SP treatment. Collectively, our results indicate that miR-34b/c-5p induced cell cycle arrest at the G2/M phase mainly by regulating CDC23 and that NK1R mainly regulates cyclin B1 and CDC23.

| MiR-34b/c-5p and NK1R regulate breast cancer cell apoptosis
We investigated the apoptosis induced by miR-34b/c-5p or NK1R silencing in MDA-MB-231 and MCF-7 cells. Annexin V/PI staining showed that transfection with pre-miR-34b/c-5p or NK1R-siRNA induced cell apoptosis that could be recovered by SP treatment ( Figure 5A). Additionally, the apoptosis rate increased as the aprepitant concentration increased (P < 0.001, Figure S5D).
This finding was further supported by our analysis of caspase-3, caspase-8 and caspase-9 activity. Transfection with pre-miR-34b/c-5p or NK1R-siRNA or aprepitant treatment significantly increased caspase-3, caspase-8 and caspase-9 activity in either F I G U R E 6 Overexpression of miR-34b/c-5p and knockdown of NK1R inhibit the tumorigenicity of breast cancer cells in vivo. A, Representative images of tumour growth. B, Tumour volume growth curves. C, Mean tumour masses 24 days after inoculation. All data are presented as the mean ± SD. D, Proteins extracted from tumour tissues were analysed by IB using anti-NK1R antibody, the densitometric evaluation of NK1R-Tr was under the band. E, The immunohistochemical stain for Ki67 is shown (×400, top). Quantification of Ki67 expression (bottom). F, Proposed regulation model of miR-34b/c-5p and SP-NK1R on cell proliferation and apoptosis in breast cancer. **P < 0.01; ***P < 0.001 MDA-MB-231 or MCF-7 cells ( Figure 5B, Figure S5E), suggesting the activation of both extrinsic and intrinsic pathways for apoptosis; however, SP treatment reduced the activity of caspase-3, caspase-8 and caspase-9 in cells transfected with pre-miR-34b/c-5p or siRNA-NK1R ( Figure 5B). The induction of apoptosis by miR-34b/c-5p or NK1R-silencing occurred through an intrinsic apoptosis pathway.

| Transfection of miR-34b/c-5p or downregulation of NK1R inhibits the tumorigenicity of breast cancer cells in vivo
Because the overexpression of miR-34b/c-5p or NK1R-silencing inhibited breast cancer cell growth in vitro, we proceeded to evaluate their effects on tumour formation in vivo. MDA-MB-231 cells overexpressing miR-34b/c-5p, NK1R silencing or their controls were inoculated into nude mice. As shown in Figure 6A and B, tumours transfected with the miR-34b/c-5p agomir grew significantly slower than control tumours (P < 0.01), and NK1R-silencing xenografted tumours grew much slower than corresponding controls (P < 0.01), which was consistent with our in vitro cell proliferation results.
Almost four weeks later, the weights of the tumours in mice transplanted with cells transfected with miR-34b/c-5p or NK1R-shRNA were significantly reduced compared with mice transplanted with control cells (P < 0.01, Figure 6C). In addition, SP treatment recovered the inhibition of overexpression of miR-34b/c-5p or NK1R knockdown on tumour size and tumour weight. We also examined Ki67 and NK1R expression in xenografted tumours. As shown in Figure 6D and E, NK1R-Tr expression and immunohistochemistry staining of the proliferation marker Ki67 were significantly downregulated following miR-34b/c-5p overexpression or NK1R knockdown. Consistent with the in vitro results, SP was able to rescue the expression inhibition of NK1R-Tr and Ki67 by miR-34b/c-5p overexpression or NK1R knockdown in vivo. These results suggest that miR-34b/c-5p and NK1R play an important role in reducing the tumorigenicity of breast cancer cells in vivo. We also observed high expression of NK1R-Tr in ER-or PR-positive patients, which was consistent with previous data. 35,38,39 Moreover, a Pearson correlation analysis showed a significant negative correlation between miR-34b/c-5p and NK1R-Tr expression in breast cancer patients, which provides further evidence for the regulation of NK1R-Tr by miR-34b/c-5p. In humans, miR-34 s are encoded by two different genes: miR-34a is encoded by its own transcript and miR-34b and miR-34c share a common primary transcript. 40,41 In this study, the close association of miR-34b-5p and miR-34c-5p is consistent with the current hypothesis that miRNAs in the same chromosomal position play a similar role in tumorigenesis. Interestingly, miR-34b-5p and miR-34c-5p reduced NK1R-FL expression by binding to the 3'UTR of NK1R-FL in MDA-MB-231, SK-BR-3, T47D

| D ISCUSS I ON
and HEK-293 T cells. These findings provide strong evidence that miR-34b-5p, miR-34c-5p and NK1R play important roles in the progression of breast cancer; thus, the roles of NK1R-Tr and NK1R-FL in breast cancer proliferation were also investigated. Moreover, aprepitant treatment also had this inhibitory effect.
Consistent with other reports, the upregulation of miR-34b/c-5p induced G2-M-phase arrest. 37 The downregulation of NK1R and aprepitant treatment also induced G2-M-phase arrest, while, SP treatment did not. Furthermore, the proliferation mechanism of miR-34b/c-5p and NK1R was tightly linked to the regulation of cell cycle regulators. CDC23, among others, is a direct target of miR-34b/c, 37,44 and our results confirmed that miR-34b/c-5p reduced its expression level in breast cancer. NK1R silencing downregulated the mRNA and protein expression of both cyclin B1 and CDC23 and SP treatment impaired the effect, which indicates that miR-34b/c-5p and NK1R arrest the cell cycle through two different signalling pathways.
Consistent with the inhibitory effects of miR-34b/c-5p overexpression and NK1R silencing on breast cancer cell growth, their ability to induce apoptosis was also shown in our Annexin V analysis.
As expected, aprepitant treatment had a similar effect on inducing cell apoptosis, well SP had the opposite effect. Furthermore, the overexpression of miR-34b/c-5p or NK1R silencing in MDA-MB-231 and MCF-7 cells increased the activity of all 3 caspases (caspase-3 effectors, caspase-8 and caspase-9 initiators), while SP treatment reversed their effect, which suggests that the induction of apoptosis by miR-34b/c-5p or NK1R not only occurs via the intrinsic pathway but also occurs extrinsically. In addition, aprepitant treatment also increased caspase-3, caspase-8 and caspase-9 activities, suggesting that extrinsic apoptosis may occur while intrinsic might be secondary.
In line with the in vitro data, our in vivo data also demonstrated that overexpression of miR-34b/c-5p resulted in a significant decrease in expression of NK1R-Tr and Ki67, as well as an inhibition of tumour growth, and SP was able to rescue the inhibition effect. Knockdown of NK1R resulted in a significant decrease in Ki67 expression, as well as an inhibition of tumour growth. Interestingly, SP could also rescue this inhibition partly.
Based on our previous results and other studies, 23,45-48 SP may rescue this effect through regulating some intermediaries positively, such as TGF-β1, EGFR and HER-2. Together, our data suggest that miR-34b/c-5p and NK1R may be potential targets for breast cancer therapy.
Importantly, this study showed that the impact of NK1R suppression on the growth and apoptosis of MDA-MB-231 cells was the same as that in MCF-7 cells, which overexpress NK1R-Tr These data suggest that the regulation of NK1R-Tr by miR-34b/c-5p may be the main mechanism for inhibiting breast cancer cell growth.
In summary, our data suggest that miR-34b/c-5p is a powerful tumour suppressor. Furthermore, the inhibition of breast cancer cell growth by miR-34b/c-5p is mainly achieved by targeting NK1R-Tr ( Figure 6F), but the specific regulatory mechanism needs further study.

ACK N OWLED G EM ENT
This work was supported by research grants from the National Natural Science Foundation of China (no. 81502519 and no. 81201653) and the Natural Science Foundation of Tianjin (no. 16JCYBJC26000).

CO N FLI C T O F I NTE R E S T
The authors declare no competing financial interests or conflicts in relation to the work described.