The miR-124-3p/Neuropilin-1 Axis Contributes to the Proliferation and Metastasis of Triple-Negative Breast Cancer Cells and Co-Activates the TGF-β Pathway

Triple-negative breast cancer (TNBC) accounts for 90% of breast cancer-associated mortality. Neuropilin-1 (NRP-1) acts as a non-tyrosine kinase receptor for several cellular signaling pathways involved in the proliferation and metastasis of cancer cells. However, the miRNAs that regulate NRP-1 expression and the underlying mechanisms in TNBC cells remain unclear. In the present study, we found that TNBC cells expressed higher levels of NRP-1 than non-TNBC cells. Stable transfectants depleted of NRP-1 were generated from two TNBC cell lines, human MDA-MB-231 and mouse 4T1 cells. NRP-1 depletion significantly suppressed the proliferation of TNBC cells by arresting the cell cycle at phase G0/G1 by upregulating p27 and downregulating cyclin E and cyclin-dependent kinase 2. NRP-1 depletion also repressed cell migration and epithelial-mesenchymal transition (EMT) by inducing the upregulation of E-cadherin and the downregulation of N-cadherin, matrix metalloproteinase (MMP)-2 and MMP-9, and reducing MMP-2 and MMP-9 activities as detected by gelatin zymography assay. By applying multiple miRNA-target prediction tools, we screened potential miRNAs with binding sites with the 3’-untranslated region of the NRP-1 gene and selected 12 miRNA candidates, among which miR-124-3p displayed the most vigorous activity to downregulate NRP-1 as validated by luciferase assay and miRNA transfection assay. By downregulating NRP-1, miR-124-3p mimics inhibited the proliferation, migration, and invasion of TNBC cells, and antagomiR-124-3p could partially abolish the effects of NRP-1 depletion. In the animal experiments, NRP-1 depletion inhibited tumorigenesis and liver metastasis of TNBC cells, while miR-124-3p mimics inhibited the growth of established TNBC tumors. In the mechanistic exploration, we revealed that NRP-1 co-interacted with transforming growth factor (TGF)-β to activate the TGF-β pathway, which regulates EMT-related molecules. In summary, the present results indicate that the miR-124-3p/NRP-1 axis contributes to the proliferation and metastasis of TNBC cells and co-activates the TGF-β pathway, suggesting that these molecules may present as potential therapeutic targets and valuable biomarkers for TNBC.


INTRODUCTION
Breast cancer is the second most common cause of cancerrelated deaths and remains a massive health burden for females worldwide (1). In particular, a subtype of breast cancer, triple-negative breast cancer (TNBC), lacks the expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2 (HER-2) and accounts for 15% of all breast cancer cases (2). It is characterized by earlier recurrence, the tendency of metastasis, shortage of effective therapeutic drugs, and poor prognosis (2,3). Despite the successful development of therapeutics for other subtypes of breast cancer, challenges remain in the management of TNBC. Therefore, it is needed to explore the molecular mechanisms accounting for the progression of TNBC.
MicroRNAs (miRNAs) are a type of non-coding RNAs containing 19-25 nucleotides and regulate over 60% of genes involved in a wide range of biological processes (17). Increasing evidence indicates that miRNAs are associated with the progression and metastasis of breast cancer (18,19). For instance, miR-10b is highly expressed in metastatic breast cancer cells and positively regulates cell migration and invasion by downregulating homeobox D10 (20). Several miRNAs have been demonstrated to regulate NRP-1 expression and participate in the progression of various types of cancer (7,(21)(22)(23). However, it's unknown what upstream mRNAs regulate NRP-1 expression in breast cancer, especially TNBC. We, therefore, designed this study to seek potential miRNAs and investigate the underlying mechanisms contributing to the proliferation and metastasis of TNBC cells. . 4T1 cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Shanghai, China), supplemented with 10% FBS (fetal bovine serum) in a humidified atmosphere of 5% CO 2 . The cell lines had been authenticated to be negative for mycoplasma infection with a short tandem repeat analysis using a PCR-based Universal Mycoplasma Detection kit (American Type Culture Collection, Manassas, VA, USA). The detailed information for antibodies, reagents, and kits is shown in Table S1 in Supplementary Material.

Luciferase Assay and Plasmid Constructs
The full-length of the 3'-untranslated region (UTR) of human NRP1 mRNA (NCBI Gene ID: 8829) was cloned into a pMIR-REPORT luciferase reporter vector (Ambion) to generate an SV40 promoter-driving luciferase reporter vector, which was used as a wild-type vector for miR-124-3p because it contained the putative miR-124-3p binding sequence (CUAUGUCCUCUC AAGUGCCUUUUUG). This binding sequence was mutated to "CUAUGUCCUCUCAACACGGAAUUUG" to generate a mutated vector. Luciferase reporter transfection and assay were performed as described previously (7). Briefly, the above vectors and an empty vector without 3'-UTR of NRP-1 were transfected into cells. Luciferase activities in cells were measured by using a luciferase assay kit (Promega, Madison, WI), and miRNA function was expressed as the percentage of the luciferase activity of the reporter vector with 3'-UTR of NRP-1 over that of the empty vector.

Animal Experiments
Three sets of animal experiments were carried out to examine the role of NRP-1 and/or miR-124-3p on the growth of TNBC tumors and liver metastasis.

Tumorigenesis Study
Two groups (n=6) of six BALB/c-nu/nu mice received a subcutaneous injection of MDA-MB-231-Sc or MDA-MB-231-NRP low cells (5 × 10 6 ) into the flank, respectively. Animals and their palpable tumors were monitored for 4 weeks, and then animals were euthanized and tumors harvested, weighed, imaged, and analyzed.

Therapeutic Effects of miR-124-3p
MDA-MB-231 cells (5 × 10 6 ) were injected subcutaneously into the flank of BALB/c-nu/nu mice. Around 10 days later, palpable tumors reached~100 mm 3 in volume and mice were randomly assigned to 3 groups (n=8), in which intratumoral injections of vehicle, negative control oligonucleotides, or miR-124-3p mimics were given, respectively. The vehicle was prepared by mixing an equal volume of serum-free medium and Lipofectamine2000 and was used to prepare transfection solution with oligonucleotides. Each tumor received a 50µl of injection solution containing 200µg of oligonucleotides. Three days after injection, 2 mice in each group were sacrificed and tumors were removed for detecting gene expression. The remaining tumors received injections on days 5, 10, and 15, and were measured every four days. The mice were monitored and euthanized on day 20.

Liver Metastasis Study
4T1-Sc or 4T1-NRP low cells (4 × 10 4 suspended in 50 µl of PBS) were injected into the left inguinal mammary fat pad of BALB/c mice, which were randomly divided into two groups (n=8). Mice were monitored for 35 days and then euthanized. The liver was harvested and fixed with 4% paraformaldehyde and transverse sections were prepared at 5 different levels to cover the entire liver. The sections were stained with hematoxylin and eosin (HE), metastatic nodules containing more than 6 tumor cells were counted, and the mean number of nodules was recorded as the number of metastases.

Other Analyses and Assays
All the other analyses and assays used in the study have been reported previously (6,23,25,26), and are described in detail in the Supplementary Material.

Statistical Analysis
GraphPad Prism 8.02 (GraphPad Software, San Diego, CA, USA) was employed for statistical analyses. Data are expressed as mean values ± standard deviation. Multiple comparisons were made with a one-way analysis of variance (ANOVA) followed by a Tukey post-hoc test. Comparisons between two groups were made by a t-test. The relationship between the expression of NRP-1 and miR-124-3p was analyzed by using Pearson's correlation coefficient. P < 0.05 was considered statistically significant.

NRP-1 Promotes the Proliferation of TNBC Cells
MDA-MB-231-NRP low cells had significantly lower viability, but MDA-MB-231-Sc cells had similar viability, compared with MDA-MB-231 cells (Figure 2A). The depletion of NRP-1 significantly downregulated the expression of cyclin E and cyclin-dependent kinase 2 (CDK2), but not had little effect on cyclin D1, in agreement with the previous studies (6,7). For further exploring the upstream factors that regulate cyclin E and CDK2, we found that NRP-1 depletion significantly upregulated the expression of p27 but not p21 ( Figure 2B). Since cyclin E and CDK2 are key regulators for cell cycle, we examined cell cycle distribution by using flow cytometry, which showed that 60.21% of MDA-MB-231-NRP low cells were arrested at the G0/G1 phase, which was significantly higher than that in MDA-MB-231-Sc cells (40.73%) ( Figures 2C, D). The BrdU incorporation assay showed that depletion of NRP-1 significantly inhibited cell proliferation as 31.6% of MDA-MB-231-NRP low cells were BrdU-positive, which was significantly lower than that of MDA-MB-231-Sc cells (78.2%) ( Figures 2E, F).

Seeking and Identifying Potential miRNAs that Regulate NRP-1 in TNBC Cells
We next screened potential miRNAs that have putative binding sites with the 3'UTR of human NRP-1 gene by using multiple miRNA prediction tools including miRanda (https://omictools. com/miranda-tool), TargetScan (http://www.targetscan.org/), miRWalk (http://mirwalk.umm.uni-heidelberg.de/), mirdb (http://mirdb.org/), and miRTarBase (http://mirtarbase.mbc. nctu.edu.tw/), which identified 12 miRNA candidates ( Figure S1 in Supplementary Material). The full-length of 3'UTR of human NRP1 mRNA (NCBI Gene ID: 8829) was inserted into an SV40 promoter-driving luciferase reporter vector, which was co-transfected into MDA-MB-231 cells with each miRNA mimics or negative control oligonucleotides ( Table S2 in Supplementary Material). Among the 12 miRNAs, miR-124-3p displayed the strongest ability to inhibit the luciferase activity ( Figure 3A). A further investigation showed a highly conserved miR-124-3p binding site on the 3'UTR of NRP1 in all the available species ( Figure 3B) and thus miR-124-3p was selected for further investigation. A mutated luciferase reporter vector was constructed by inserting a mutated 3'-UTR of NRP-1 gene ( Figure 3C). The inhibitory effect of miR-124-3p on the luciferase activity was shown to be in a dose-dependent manner ( Figure 3D). MiR-124-3p mimics or antagomiR-124-3p significantly altered the luciferase activity in cells co-transfected with the wild-type vector but not the mutated vector ( Figure 3E). The results indicate that the binding site on NRP-13'-UTR is essential for miR-124-3p to display its regulatory effect in MDA-MB-231 cells.
By applying qRT-PCR analyses, we detected the expression of miR-124-3p in available cell lines and found that MDA-MB-231 cells expressed the lowest level of miR-124-3p ( Figure S2A Figure 5A and Figure S3 in Supplementary Material). Similar results were obtained in mouse 4T1 cells ( Figure S4 in Supplementary Material). In mechanistic exploration, the role of miR-124-3p on cell proliferation was supported by its regulatory effects on the expression of CDK2, cyclin E and p27 through downregulating NRP-1 expression ( Figure 5B). Specifically, miR-124-3p mimics downregulated the expression of cyclin E and CDK2, and upregulated the expression of p27, while antagomiR-124-3p showed an opposite effect ( Figure 5B). Since antagomiR-124-3p inhibits the negative regulatory function of miR-124-3p, which binds to the 3'-UTR of NRP-1 mRNA, while NRP-1 shRNA targets the coding sequence (CDS) of NRP-1 mRNA, two regulatory mechanisms on NRP-1 expression exist in MDA-MB-231-NRP low cells transfected with antagomiR-124-3p. In addition, NRP-1 shRNA and antagomiR-124-3p may also transfect different cells because they are unable to transfect 100% of cells. Therefore, antagomiR-124-3p only partially abolished the effects of NRP-1 depletion ( Figure 5B).   Figures  6A, B) and scratch ( Figures 6C, D) assays. It is well known that breast cancer cells gain their ability to migrate and invade through the epithelial-to-mesenchymal transition (EMT) (27). We showed here that NRP-1 depletion led to significantly downregulation of N-cadherin, matrix metalloproteinase (MMP)-2 and MMP-9, and upregulation of E-cadherin ( Figure 6E). The results of gelatin zymography assays also showed that MDA-MB-231-NRP low cells had lower activities of MMP-2 and MMP-9 ( Figure 6F). Furthermore, miR-124-3p mimics reduced the number of migrated cells, while antagomiR-124-3p partially abolished the effect of NRP-1 depletion on cell migration ( Figure 6G); and similar results were obtained with mouse 4T1 cells ( Figure S4 in Supplementary Material). The above effects of miR-124-3p were in accordance with the changes in E-cadherin and N-cadherin expression ( Figure 6H) and the results of Matrigel invasion assay ( Figure S5 in Supplementary Material).  Figure 7B). In agreement with NRP-1 expression in cultured cells ( Figure 1D and Figure 2B), MDA-MB-231-NRP low tumors had a weaker expression of NRP-1 as examined by immunohistochemistry ( Figure 7C). MDA-MB-231-NRP low tumors also had weaker cell proliferation detected by immunohistochemistry with an Ab against Ki-67 ( Figures 7C, D). The effects of NRP-1 depletion on liver metastasis of TNBC cells were examined by adopting mouse 4T1 cells, which can A one-way ANOVA with a Tukey post-hoc test was used for statistical analysis. "**P < 0.001" indicates a significant difference; " f P < 0.05" and " ff P < 0.001" indicate a significant increase, while " # P < 0.05" and " ## P < 0.001", a significant reduction, compared with negative control-transfected cells. "*" (P<0.05) and "**" (P < 0.001) indicate a significant difference. A one-way ANOVA with a Tukey post-hoc test was used for statistical analysis. " f P < 0.05" indicates a significant increase, while " # P < 0.05", a significant reduction, compared with negative control-treated MDA-MB-231-Sc cells. spontaneously metastasize from the primary tumors to distant sites including livers (28). Primary 4T1-NRP low tumors were significantly smaller than control 4T1-Sc tumors ( Figure S6 Figure 8A). The downregulation of NRP-1 and reduced expression of Ki-67 in miR-124-3p mimics-treated tumors harvested 3 days after gene injection were confirmed by immunohistochemistry ( Figures  8B-D). Western blot analysis of tumor homogenates also showed downregulation of NRP-1, cyclin E, CDK2 and Ncadherin, and upregulation of p27 and E-cadherin in miR-124-3p mimics-treated tumors ( Figure 8E).

NRP-1 Co-Activates the TGF-b Signaling Pathway Through Binding With TGF-b
It has been reported that NRP-1 can co-activate the TGF-b pathway (29), which plays a key role in the progression of TNBC (30). We confirmed the binding of TGF-b and NRP-1 in MDA-MB-231 cells as detected by co-immunoprecipitation assay ( Figure S7 in Supplementary Material), in agreement with published studies (8,31). We also showed that the interaction protein, a stimulator of the TGF-b pathway, and galunisertib, which is a specific inhibitor of TGF-b receptor (TGF-bR) and has been tested to treat breast cancer in clinical trials (32), or their combination were added to cultured cells. TGF-b protein and/or galunisertib had little effect on the expression of NRP-1 or TGF-bRI ( Figure 9A). However, recombinant TGF-b protein increased, while galunisertib reduced, the expression of p-TGF-bRI. The activation of the TGF-b signaling pathway by TGF-b protein, or the inhibition by galunisertib or NRP-1 depletion, had no effect on the expression of Smad2/3 or Samd4. However, TGF-b protein incubation induced an increase in p-Smad2/3, Snail and N-cadherin expressions, and reduced the expression of E-cadherin; while inhibition of TGF-b signaling by galunisertib or NRP-1 depletion reduced the expression of p-Smad2/3, Snail and N-cadherin, and increased the expression of E-cadherin, and also abolished the effects of TGF-b protein in part ( Figure 9A).

DISCUSSION
NRP-1 is involved in the progression of various cancer types by co-activating multiple cognate receptor tyrosine kinase signaling pathways (6,7,23). In breast cancer, particularly TNBC, NRP-1 is an isoform-specific receptor for VEGF and the VEGF/NRP-1 axis promotes cell proliferation and migration by increasing the activity of cell division control protein 42 homolog (Cdc42) (14) or enhancing the EMT and activation of NF-kB and b-catenin (15). In accord, the present study has demonstrated that NRP-1 acts as a co-receptor for TGF-b to activate the TGF-b pathway in TNBC cells. We have also identified miR-124-3p as an upstream regulator for NRP-1 by binding to its 3'-UTR in TNBC cells ( Figure 9B). Some miRNAs, such as miRNA-148, miR-124, miR-320 and miR-141, have been shown to negatively regulate the expression of NRP-1 in hepatocellular carcinoma (HCC) (21), glioma (22), cholangiocarcinoma (23) and pancreatic ductal carcinoma (7), respectively, supporting the theory that one target gene can be regulated by multiple miRNAs (17). To our knowledge, the present study may be the first one revealing a regulatory link between miR-124-3p and NRP-1 and elucidating some of the functional role of the miR-124-3p/NRP-1 axis in the proliferation and metastasis of TNBC cells.
MiR-124 is one of the most studied miRNAs because it is downregulated and contributes to the development, progression and prognosis in various human malignancies (33). In a study with miRNA library screening with functional proteomics and integrated analysis of clinical data, miR-124 has been identified to be a potential suppressor capable of reducing p27 expression by binding to the promoter region, leading to a subsequent G1 arrest and inhibiting the invasive ability of breast cancer cells (18). In the present study, we have demonstrated that miR-124-3p reduced the expression of p27 through downregulating NRP-1. It has been reported that NRP-1, by co-activating the HGF/c-Met pathway (6), increases the phosphorylation of Akt (34), which in turn leads to p27 downregulation, inhibiting the activation of the CDK2/cyclin E complex, resulting in sequential cell cycle arrest (35). However, other studies suggest that miR-124-3p exerts a suppressive function by targeting different genes. For example, miR-124 downregulates PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha), suppressing the proliferation of HCC cells (36); miR-124-3p inhibits the invasiveness and metastasis of HCC cells by targeting CRKL (Crk-like protein) (37); and miR-124-3p regulates FGF2 (fibroblast growth factor 2)-EGFR pathway to overcome pemetrexed resistance in lung adenocarcinoma cells by targeting MGAT5 (alpha-1,6-mannosylglycoprotein 6-beta-Nacetylglucosaminyltransferase) (38). These results suggest that the role of miR-124-3p is complex, and cell-and disease-dependent.
pathway and promote EMT in TNBC cells ( Figure 9B). In support, it has been reported that NRP-1 serving as a highaffinity receptor for TGF-bcan activate the TGF-b signaling in MDA-MB-231 cells (8,31). Interestingly, NRP-1 has also been reported to contribute to TGF−b−induced EMT and metastasis of non−small cell lung cancer cells by binding with TGFbRII (40). In the TGF−b signaling pathway, TGF−b binds to TGFbRII on the cell membrane to recruit TGFbRI and form a complex, leading to the phosphorylation of TGFbRI (41). On the other hand, NRP-1 depletion or inhibition of TGF-b signaling by galunisertib could inhibit the phosphorylation of TGFbRI ( Figure 9B). Therefore, the interaction of NRP-1 with the TGF-b pathway may be conducted through binding with different parts of the TGF−b complex in different cell types.
The phosphorylated TGFbRI can, in turn, induce the phosphorylation of Smad2/3 that forms a trimeric SMAD with Smad4, leading to the upregulation of Snail, which conveys TGFb-induced repression of N-cadherin upregulation and Ecadherin downregulation, the two well-known hallmarks of EMT (41) ( Figure 9B). EMT is also associated with the increased expression and activities of MMP-2 and MMP-9, which can stimulate the metastasis of cancer cells (42).
Although the regulatory effect of miR-124-3p on NRP-1 expression and their roles on the proliferation and metastasis of human and mouse TNBC cells have been evaluated in the study, the molecular expression and interaction have not been fully validated in mouse 4T1 cells due to the unavailability of specific antibodies. Further investigations on this point by adopting mouse TNBC cells, and maybe other human cell lines, can demonstrate the reproducibility in phenotypes and confirm the generalizability of the miR-124-3p/NRP-1 axis in TNBC cells.
We have previously reported that the miR-141/NRP-1 axis was associated with clinicopathology and contributed to the growth and metastasis of pancreatic cancer (7). Unfortunately, we have not validated the significance of the miR-124-3p/NRP-1 axis in clinical TNBC data. Such a study may further confirm the role of this axis by analyzing the association of expression levels of miR-124-3p and NRP-1 with clinicopathological parameters and survival data of human TNBC. It has been reported that the expression level of NRP-1 correlates with lymph metastasis (13,15) and inversely correlates with the survival in breast cancer (16). A recent study suggests that NRP-1 may be an independent prognostic factor for TNBC patients (43) and increased NRP-1 expression has been observed after neoadjuvant chemotherapy in breast cancer patients (44). All these studies highlight the significance and importance of NRP-1 in breast cancer, particularly, TNBC.
TNBC cells, such as MDA-MB-231 and MDA-MB-453 cells, have been shown to express higher levels of NRP-1 than non-TNBC MCF-7 and ZR-75-1 cells (45,46). The present study has confirmed this finding that TNBC cells expressed higher levels of NRP-1 and lower levels of miR-124-3p than non-TNBC cells. The higher expression of NRP-1 has also been found in TNBC tumor tissues (16). TNBC is characterized by lacking the expression of estrogen receptor, progesterone receptor, and HER-2 (2). Therefore, the intrinsic molecular linkage of these three receptors, particularly, HER-2, with the miR-124-3p/NRP-1 axis may be worth further investigation for elucidating the underlying mechanisms.
In summary, TNBC only accounts for around 15% of all breast cancers but it is responsible for about 90% of breast cancer-associated mortality (2,3). The lack of effective therapeutics highlights the need for further exploring the underlying mechanisms contributing to its aggressive features. The function of the miR-124-3p/NRP-1 axis involved in the proliferation and metastasis of TNBC cells suggests that these molecules may be potential therapeutic targets and valuable biomarkers for TNBC and warrant further investigation.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The animal study was reviewed and approved by the Animal Ethics Committee of Harbin Medical Unversity.

AUTHOR CONTRIBUTIONS
XS and XJ designed the study. JZ and XZ performed the majority of the experiments and data analysis. ZL and YS assisted with the in vivo experiments. JZ and XS drafted the manuscript. JZ and XZ contributed equally to this work. All authors contributed to the article and approved the submitted version.