M2 macrophages contribute to cell proliferation and migration of breast cancer

Breast cancer is a kind of malignant tumor that severely threatens women's lives and health worldwide. Tumor‐associated macrophages (TAMs) have been reported to mediate tumor progression, while the mechanism still needs further identification. In this study, we found that M2 macrophages promoted increased cell proliferation and migration as well as reduced expression of interferon regulatory factor 7 (IRF7) and increased the expression of miR‐1587 in breast cancer cells. Overexpression of IRF7 or miR‐1587 knockdown reversed M2 macrophage‐induced cell proliferation and migration as well as tumor growth in vivo. Mechanistically, miR‐1587 targeted the 3ʹ‐untranslated region (3ʹ‐UTR) of IRF7 mRNA to regulate its protein expression leading to tumor progression. Collectively, this study revealed that the miR‐1587/IRF7 axis mediates M2 macrophage‐induced breast cancer progression, and this sheds light on further clinical therapy for breast cancer by targeting TAMs as well as the miR‐1587/IRF7 axis.


| INTRODUCTION
Breast cancer is the most common cancer in women and the second leading cause of cancer-related death. Annually, more than 1.8 million women have been diagnosed with breast cancer worldwide, and most of them had no metastatic disease, resulting in poor prognosis and a high mortality rate (Bray et al., 2018). The biological behaviors of breast cancer are early recurrence, rapid malignant progression, as well as a short overall survival time, which seriously threaten patients' lives and health. Despite the improvement of detection and treatment techniques, the mortality rate is still high. Therefore, understanding the pathology of breast cancer progression would be important for reducing the mortality rate and increasing patients' survival.
The tumor microenvironment (TME) consists of a complex interaction among various cells such as tumor cells, immune cells, endothelial cells, and fibroblasts. Tumor-associated macrophages (TAMs), accounting for 50%-80% of interstitial cells, are important components of the TME . TAMs mediate immune response by inhibiting T-cell proliferation and preventing expression of immunosuppressive molecules, which could eliminate tumor cells, and promote tumor growth and survival (Aras & Zaidi, 2017;Zhao et al., 2017). In addition, TAMs are correlated with a poor prognosis of breast cancer (Choi et al., 2018). TAMs can be classified into classical pathwayactivated macrophages (M1-type) and alternative pathwayactivated macrophages (M2-type). M1-type TAMs mainly show an anticancer effect, while M2-type mainly manifest the opposite (Mantovani et al., 2017). TAMs in tumor tissues tend to polarize into the M2 phenotype, which produces anti-inflammatory factors, and contributes to tumor cell immune escape, further promoting tumor progression. Therefore, identifying the mechanism of M2 on breast cancer progression seems to be important.
Type I interferons (IFNs) are the main defensive factors of the innate immune response during the viral infection, and thus identified as therapeutic agents (Alberti & Benvegnu, 2003;Festi et al., 2004). The expression of IFNs is mediated by the pattern recognition receptors and further leads to the phosphorylation of IFN regulatory factor 7 (IRF7) and IRF3 (Akira et al., 2006). It has been reported that IRF7 activation and expression could be regulated by various mechanisms at the transcriptional, translational, and posttranslational levels (Ning et al., 2011). Moreover, the IRF7 activity and stability are also regulated by its phosphorylation, ubiquitination, SUMOylation, and acetylation (Ning et al., 2011). In addition, IRF7 is identified to mediate cancer progression and inflammatory disorders (Ling et al., 2019;Rajagopalan et al., 2018).
And the progression of breast cancer is controlled by IRF7 (Lan et al., 2019;Touati et al., 2017).
In this study, we found that M2 could suppress IRF7 expression, which promotes cell proliferation and migration. Afterwards, the potential mechanism underlying IRF7-mediated breast cancer progression was explored. We showed that miR-1587 targets IRF7 to regulate M2-induced breast cancer progression.

| Cell culture
The human monocytic cell line THP-1 and human breast cancer cell line MDA-MB-231 and BT-549 were purchased from the American Type Culture Collection and cultured in Dulbecco's modified Eagles' medium supplemented with 10% fetal bovine serum at 37°C with 5% CO 2 . To induce M2-type macrophages, THP-1 were pretreated with phorbol 12-myristate 13-acetate (PMA, 100 ng/ml) for 18 h and then cultured with interleukin-4 (IL-4; 25 ng/ml) for 3 days. For the following experiments, both M2-type macrophages and THP-1 cells were implanted into the 96-well plates with 2 × 10 5 cells/ml, 200 µl/well. After 24 h, the supernatant (100 μl) was collected to culture MDA-MB-231 cells for 24 h.

| Real-time polymerase chain reaction (PCR)
Total RNA was isolated from cells using TRIzol reagent (Invitrogen) and quantified by 10% agarose gel electrophoresis. The extracted RNA was used to reverse transcribe into complementary DNA (cDNA). For realtime PCR, cDNA was used as the template, and SYBR Green Real-time PCR Master Mix (Toyobo) was used for the experiments based on an ABI 7500 real-time PCR system (Applied Biosystems). The mRNA expression was assessed using the 2 C t -ΔΔ method. GAPDH served as internal control. The primers sequences used in this study are as follows: IRF7, Forward, 5ʹ-AAACCATAGAGGCACCCAAG-3ʹ; Reverse, 5ʹ-TTGGGAGTTGGGATTCTGAGTCAAGGC-3ʹ; GAPDH, Forward: 5ʹ-ACCACAGTCCATGCCATCAC-3ʹ; Reverse, 5ʹ-TCCACCACCCTGTTGCTGTA-3ʹ.

| Western blot analysis
The cells were treated by radioimmunoprecipitation assay (

| Cell proliferation
Cell proliferation was determined using a Cell Counting Kit-8 (CCK-8, Sangon Biotech). Briefly, cells were implanted into a 96-well plate with a concentration of 10 3 cells/well. After 24 h, the cells were incubated with CCK-8 (10 μl) solution at 37°C for 3 h. A microplate reader was employed to detect the absorbance at 450 nm (Bio-Rad).

| Cell migration
For determination of cell migration, scratch wound healing assays were performed. Briefly, cells were seeded in a 6-well plate with a concentration at 10 5 cells/well. After 25 h, the cells were spread across the plate and a 200 μl micropipette tip was used to scratch through the cells. After washing by PBS, the cells were cultured at 37°C with 5% CO 2 . After 24 h, cell migration was photographed with an inverted microscope (×40, Olympus).
After reaching 70% influence, the cells were transfected with the Lipofectamine 3000 (Invitrogen) according to the manufacturer's instruction. After 24 h, the transfection efficiency was determined.

| Dual-luciferase reporter assay
The fragment of IRF7 3ʹ-UTR containing the predicted binding sites of miR-1587 wild-type (WT) or mutant (MUT) was amplified and cloned into pmirGLO luciferase vector (Promega). Next, the miR-1587 mimic or its negative control was cotransfected with the reporter plasmid into HEK293 cells. After 48 h, the luciferase activity was determined using a dual-luciferase reporter assay system (Promega) following the manufacturer's instruction.

| Statistical analysis
All experiments were repeated three independent times. Statistical analysis was performed using SPSS 20.0 and GraphPad prism 8.0 software. Data were presented as means ± SD. Statistical difference between the two groups was analyzed using Student's t test, while one-way analysis was conducted to determine the difference among three or more groups. p < .05 was considered as a statistically significant difference.

| The increased miR-1587 expression promotes cell proliferation and migration
To explore the potential mechanism of IRF7 on breast cancer progression, we selected potential IRF7-interacting microRNAs (miRNAs) using TargetScan. It showed that miR-1587 has a binding site with IRF7 3ʹ-UTR. miR-1587 has also been identified to mediate TU ET AL.

| IRF7 is a target of miR-1587
To identify the interaction between miR-1587 and IRF7, MDA-MB-231 cells were genetically overexpressed with miR-1587. We found that the IRF7 expression was increased by miR1587 overexpression, while knockdown of miR-1587 simultaneously suppressed IRF7 expression (Figure 4a,b). Based on an online prediction using TargetScan, we found that miR-1587 has potential binding sites with IRF7 3ʹ-UTR ( Figure 4c). Next, the luciferase reporter plasmids containing WT-IRF7 3ʹ-UTR or MUT-IRF7 3ʹ-UTR were cotransfected with miR-1587 mimic into HEK293 to determine the regulatory pattern of miR-1587 on IRF7 expression, and the results showed that miR-1587 mimic reduced the relative luciferase activity of Luciferase reporter plasmids containing WT IRF7 3ʹ-UTR, but not the mutated one ( Figure 4d). All those results demonstrated that miR-1587 targets IRF7 to suppress its expression.

| miR-1587 promotes cell progression by downregulating IRF7 in breast cancer
We further evaluated the interaction between miR-1587 and IRF7 on breast cancer progression. As is presented in the Figure 5a

| DISCUSSION
It is accepted that the TAMs contribute to tumor progression by enhancing the capacity of tumor invasion, metastasis, drugresistance, angiogenesis, as well as immune escape (Banerjee et al., 2011;Liu et al., 2017;Tevis et al., 2017). from TAM-derived exosomes promotes pancreatic ductal adenocarcinoma (Yin et al., 2019). In this study, we found miR-1587 promoted breast cancer progression by mediating TAMs.
miR-1587 function is regulated by the induction of G-quadruplex formation and dimerization (Tan et al., 2018). Glioma-associated mesenchymal stem cell-derived exosomal miR-1587 contributes to the tumorigenicity (Figueroa et al., 2017). miR-1587 targets ZEB2 to regulate cell proliferation, migration, apoptosis, and autophagy in keloid fibroblasts (Hou et al., 2019). In this study, we found that breast cancer cells treated by the supernatant medium of M2 macrophage showed a high level of miR-1587, and miR-1587 overexpression contributes to cell proliferation and migration of breast cancer cells, indicating that miR-1587 mediates TAMs function, consistent with a previous report exosomes enriched miR-1587 mediates glioma progression (Figueroa et al., 2017). We found that miR-1587 is enriched in breast cancer cells pretreated with the supernatant of M2 macrophages, and further promoted their progression. However, whether miR-1587 is enriched in the M2 macrophages still needs further identification.
Recent studies have shown that IRF1, IRF6, IRF7, and IRF8 served as cancer suppressor genes and mediated breast cancer cell activity (Luo et al., 2017;Xu et al., 2019). IRF7 is an important family member of IRFs and acts as a transcription factor to manipulate IFN expression, as well as IFN stimulating response genes. The phosphorylated IRF7 that is induced by TBK1/IKK can be transported to the nucleus and then participates in IRF3-mediated biological processes under virus infection (Chen et al., 2017). IRF7 expression has been studied in various cancers. For example, the upregulated IRF7 mediated cell migration and invasion of non-small-cell lung cancer.
Elevated IRF7 expression is associated with poor prognosis of patients with renal cell carcinoma (Lin & Cai, 2020). In addition, the downregulated IRF7 is correlated with a lower overall survival of gallbladder carcinoma (Goeppert et al., 2019). In breast cancer, upregulation of IRF7 in the treated cancer cells promoted resistance to chemotherapy (Lan et al., 2019). In this study, we found that IRF7 was decreased in the breast cancer cells that were treated by the supernatant medium of M2 macrophage, indicating that IRF7 mediated TAM function in breast cancer. Interestingly, miR-1587 has binding sites with IRF7 3ʹ-UTR, combining with the luciferase assays, we concluded that miR-1587 targets IRF7 to regulate its expression.
Breast cell behaviors such as cell proliferation, migration, invasion, and apoptosis have been considered in the study of tumor progression. In this study, we found that the supernatant medium of M2 macrophage promoted tumor progression by inducing cell proliferation and migration, which might be due to the fact that M2 could produce anti-inflammatory factors and induce immune escape.
In addition, the supernatant medium of M2 macrophage treatment