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M2 polarization induction of macrophage
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miR-200c/PAI-2 promotes the progression of triple negative breast cancer via M1/M2 polarization induction of macrophage ⁎
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Ziqi Menga, Rui Zhanga, Yixuan Wanga, Guang Zhua, Tiefeng Jina, , Chunguo Lic, , ⁎ Songnan Zhangb, a
Department of Pathology and Cancer Research Center, Yanbian University Medical College, Yanji 133002, China Department of Oncology, Yanbian University Hospital, Yanji 133002, China c Department of Oncology, Jilin City Central Hospital, Jilin 132011, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: miR-200c PAI-2 MDA-MB-231 Macrophages Cell migration Transwell co-culture
Purpose: To investigate the effect of miR-200c/PAI-2 on macrophage polarization into M2-type TAMs in TNBC. Methods and materials: PAI-2 expression in MDA-MB-231con, MDA-MB-231miR-200ab and MDA-MB-231miR-200c breast cancer cells was evaluated by RT-PCR and immunofluorescence (IF), while the expression of the TAM marker F4/80 and the M2-type TAM marker CD206 in MDA-MB-231con, MDA-MB-231miR-200c and MDA-MB231miR-200c/siPAI-2 mouse lung metastatic tumor tissues was examined with immunohistochemistry (IHC). The effects of RAW264.7 cells on MDA-MB-231con, MDA-MB-231miR-200c and MDA-MB-231miR-200c/siPAI-2 were examined by transwell co-culture. CD206 expression in RAW264.7 cells were confirmed by immunostaining. The level of PAI-2 and IL-10 in the co-culture supernatants were assessed using ELISA. Results: 1. RT-PCR and IF analysis showed that PAI-2 was upregulated in MDA-MB-231miR-200c cells. 2. IHC assays analysis showed that the numbers of F4/80 and CD206 positive cells were increased in MDA-MB-231miR200c tumor tissues, while in MDA-MB-231miR-200c/siPAI-2 tumor tissues were decreased. 3. Transwell co-culture assays analysis showed that MDA-MB-231miR-200c cells significantly promoted the cell migration ability compared with the control group, while knockdown PAI-2 significantly inhibited the cell migration ability (P < 0.05). 4. Transwell co-culture and immunostaining assays analysis showed that overexpression miR-200c in MDA-MB-231 cell line increased the CD206 expression in RAW264.7 cells, while knockdown PAI-2 decreased. 5. ELISA assays analysis showed that miR-200c-mediated MDA-MB-231 cells significantly increased the secretion of PAI-2 and IL-10, while decreased the secretion of PAI-2 and IL-10 in MDA-MB-231 miR-200c/siPAI-2 cells. Conclusions: miR-200c promotes the malignant progressions of TNBC by PAI-2 upregulation and M2 phenotype macrophages polarization.
1. Introduction
important components of the tumor microenvironment, accounting for 50%-80% of interstitial cells. Studies have shown that the numbers of invasive TAMs are positively correlated with breast cancer staging, grading, metastatic potential, and prognosis [2]. Though macrophages are an important part of the body's innate immunity and play a role in tumor immunity during tumor initiation [3], macrophages are highly plastic. Under the inductions of certainfactors, the phenotypes, functions and morphologies of macrophages will be changed and finally lead to macrophage polarization [4]. TAMs can be classified into a classical pathway-activated macrophages (M1-type) and alternative pathway-activated macrophages (M2-type). M1-type TAMs mainly shows an anti-cancer effect, while M2-type TAMs mainly shows the
Breast cancer can be divided into four subtypes, luminal A, luminal B, HER2 and triple-negative breast cancer (TNBC). The biological behavior and treatment strategies of these different molecular subtypes are completely different. Patients with TNBC, due to the lack of specific molecular targets, they usually faced many problems such as early recurrence, rapid malignant progressions and a short overall survival time, the five-year overall survival rate for patients with TNBC is only 64.97% [1]. Thus, it is important to find new treatment strategies. The tumor microenvironment is composed of tumor cells, mesenchymal cells and immune cells. Tumor-associated macrophages are
⁎ Corresponding authors at: Yanbian University, 977 Park Road, Yanji City, Jilin Province, China (T. Jin). Jilin City Central Hospital, Jilin City, Jilin Province, China (C. Li). Yanbian Hospital, Yanji City, Jilin Province, China (S. Zhang). E-mail addresses: [email protected] (T. Jin), [email protected] (C. Li), [email protected] (S. Zhang).
https://doi.org/10.1016/j.intimp.2019.106028 Received 12 August 2019; Received in revised form 21 October 2019; Accepted 4 November 2019 1567-5769/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Ziqi Meng, et al., International Immunopharmacology, https://doi.org/10.1016/j.intimp.2019.106028
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Fig. 1. miR-200 overexpression upregulated PAI-2 in MDA-MB-231 cells. (A) Representative images and (B) complete data of PAI-2 RT-PCR of MDA-MB-231con and MDA-MB-231miR-200ab/c cells. (C) Immunofluorescence staining of PAI-2 in MDA-MB-231miR-200ab/c cells and MDA-MB-231con cells (×400).
2. Materials and methods
opposite [5]. Recently, many studies discovered that TAMs in tumor tissues tend to polarize into the M2 phenotype [6], and this effect mainly depends on cancer types and stromal interactions. Macrophage polarization is confirmed as a key process in cancer progression and macrophages express different states in response to tumor microenvironmental signals [7]. Choi J. et al. confirmed that M2-type TAMs promote tumor invasion, metastasis, immunosuppression and drug resistance by inducing angiogenesis, degrading the tumor extracellular matrix and helping tumor cells escape from immune regulation during the progression of breast cancer [2]. Therefore, reversing the polarization of M2-type TAMs, reducing their recruitment and blocking the tumor-promoting function of M2-type TAMs could be a new anti-tumor strategy. The miR-200 family (including miR-200a, miR-200b, miR-200c, miR-141 and miR-429) is an important group of miRNAs. It plays an important role in maintaining epithelial traits in cells or tissues and in regulates tumor proliferation, invasion and metastasis. Studies have found that the expression level of miR-200 is closely related to the prognosis of breast cancer patients [8], but there are few reports on whether it influences the TAMs polarization. This study explored the effects of miR-200 on the polarization of TAMs and the biological characteristics of TNBC, and it provides new ideas to further understand the role of miR-200 in TAMs polarization and the development of TNBC. Our previous study showed that overexpression miR-200 upregulated the expression of plasminogen activator inhibitor-2 (PAI-2) in MDA-MB-231 cells [9]. The expression of miR-200c in breast cancer cells can promote the polarization of M2-type macrophages [10]. However, the exact role of PAI-2 in TAM polarization and its underlying mechanisms remain unknown. This study aimed to clarify whether PAI-2 is involved in miR-200c-mediated M2 phenotypic polarization.
2.1. Cell culture Mouse macrophage cells RAW264.7 and human breast cancer cells MDA-MB-231 were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cell lines were cultured in RPMI1640 and DMEM medium respectively, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C, 5% CO2.
2.2. Immunohistochemistry The tissue was fixed with 10% formalin and embedded in paraffin blocks. Paraffin-embedded continuous tissue sections were used for immunohistochemical staining of F4/80 and CD206. Briefly, 4 µm tissue sections were subsequently dewaxed and rehydrated using xylene and gradient alcohol washes. Antigen retrieval was performed at 121 °C for 1 min using pH 6.0 citrate buffer. After hydrogen peroxide blocking at room temperature for 20 min, sections were incubated with primary polyclonal antibodies against F4/80 (Thermo Fisher Scientific Inc.) and CD206 (Cell Signaling Technology, 91992, USA) overnight at 4 °C. After incubation with the secondary antibody at room temperaturefor 1 h, immunostaining with DAB and counterstaining of the slide with hematoxylin, TAMs were scored as the infiltration density of F4/80+ or CD206+ cells inbrown-yellow monocyte/macrophage morphology. TAMs were evaluated by adapting the reported hotspot quantitative method. The entire stained section was first scanned at low magnification (×200) to identify the hotspot areas (five areas of tumor stroma) with the most abundant macrophages. The average count of macrophages in all five areas and the density of infiltration was estimated (per 0.24 mm2) at higher magnification (×400). Two researchers (pathologists) blinded to the information carried out the evaluation process. 2
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Fig. 2. PAI-2 knockdown decreased M2 phenotype polarization by miR-200c overexpression in MDA-MB-231 cells. (A) Representative BLI of mice injected with MDA-MB-231con, MDA-MB-231miR-200c and MDA-MB-231miR-200c/siPAI-2 cells. (B) Total photon flux of the bioluminescent signals emitted from thorax regions of MDAMB-231con, MDA-MB-231miR-200c and MDA-MB-231miR-200c/siPAI-2 mice. (C) Representative H&E staining in lung tissues. (D) Tumor area of individual lung metastases evaluated via H&E staining in MDA-MB-231con, MDA-MB-231miR-200c and MDA-MB-231miR-200c/siPAI-2 mice. (E) Representative F4/80 immunostaining in lung tissues. (F) F4/80 tumor staining area of individual lung metastases evaluated via IHC staining. (G) Representative CD206 immunostaining in lung tissues. (H) Randomly chosen staining area of CD206 positive macrophages in lung metastatic tumor around in the 5 field of view (×200).
and visualized by ethidium bromide. All assays were performed at least three times.
2.3. Real-time PCR Total RNA was isolated by use of TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed using a Prime Script™ RTPCR Kit (Takara, RR014A). Real-time PCRs reactions were run on an ABI PRISM 7900 utilizing a SYBR Green PCR master mix (Applied Biosystems, Foster City, CA, USA) and specific primer sets for PAI-2. After amplification, PCR products were separated on 1% agarose gels
2.4. Colony formation assay Cells were plated on 6-well plates at a concentration of 5 × 103 cells for each well and incubated for 12 days at 37 °C. The cells were fixed in 4% paraformaldehyde for 15 min and then stained with Giemsa for 3
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Fig. 3. RAW264.7 cells promote the migration of MDA-MB-231 cells. (A-B) Transwell migration assay of MDA-MB231con, MDA-MB-231miR-200c and MDAMB-231miR-200c/siPAI-2 for 24 h. (C-D) Transwell migration assay of 1 × 104 RAW264.7 cells that were co-cultured with MDA-MB-231con, MDA-MB231miR-200c and MDA-MB-231miR-200c/ siPAI-2 for 24 h. (E) Cell morphology image of RAW264.7 cells co-cultured MDA-MBwith MDA-MB-231con, miR-200c and MDA-MB-231miR-200c/ 231 siPAI-2 for 24 h. (F) The level of IL-10 in the co-culture supernatants was assessed using ELISA. (G) The level of PAI-2 in the co-culture supernatants was assessed using ELISA (All values from three independent experiments are quantified as the mean ± SD, P < 0.05).
2.7. Lentiviral transduction
25 min after being washed with cold PBS.
The pLent-H1-GFP-Puro-based lentiviruses carrying luciferase were purchased from Vigene Biosciences and transfected into MDA-MB-231 cells using DMEM medium containing polybrene (5 μg/ml), respectively. Lentiviral vectors containing the miR-200ab/c constructs were kindly supplied by Dr. Gregory J. Goodall. After transduction of miR200c lentivirus into MDA-MB-231 cells and selection with puromycin (3 μg/ml), MDA-MB-231 cells overexpressing miR-200ab/c were sorted using a FACS caliber flow cytometer (BD Biosciences, CA, USA). Additionally, lentivirus was transduced into miR-200ab/c-overexpressing MDA-MB-231 cells for animal studies. MDA-MB-231 cells and miR-200ab/c-overexpressing MDA-MB-231 cells are referred to as MDA-MB-231con and MDA-MB-231miR-200ab/c.
2.5. MTT assay Cells were seeded onto 96-well plates at a density of 5 × 103 cells per well and incubated for 0, 24, 48, 72 h. 100 μL medium containing 20 μL of MTT reagent (5 mg/mL) was added into each well and incubated for 4 h under the same conditions. Then, 100 μL of DMSO was added to each well and the absorbance value (OD) at 490 nm of each well was measured using a microplate reader (LabSystemsMultiskan Ascent). Five wells per group at least were analyzed and repeated three times.
2.6. Transfection 2.8. Immunofluorescence We purchased three different PAI-2 siRNA from Bioneer. siRNA sequences were as follows: (1) PAI-2 siRNA sense, AAGGUAUCCCUA UUUUUGAAGCCUGUCUC and antisense, AACUUCAAAAAUAGGGAU ACCCCUGUCUC (2) PAI-2 siRNA sense, GAGCUUCCGGGAAGAAUAU and antisense, AUAUUCUUCCCGGAAGCUC (3) PAI-2 siRNA sense, CAGAGAACAACCAGAUUGA and antisense, UCAAUCUGGUUGUUCU CUG. Scramble siRNA (AccuTarget™ Negative Control siRNA, BioRP) was also used. Cells were transfected with 30 nM PAI-2 siRNA or scramble siRNA using Lipofectamine-3000 (Invitrogen).
Cells grown in 6-well plates culture slides were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.5% Triton X-100 (CWBIO, China) and blocked with 3% BSA for 2 h. Cells were incubated with primary antibody in 3% BSA at 4 °C overnight, washed three times with PBS, incubated with Alexa Fluor 568-labeled secondary antibody (Invitrogen) in 3% BSA for 2 h, and then analyzed by Leica SP5II confocal microscope.
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Fig. 4. miR-200c/PAI-2 axis promotes the malignant progression of triple- negative breast cancer cells by inducing the polarization of macrophages into M2 direction. (A) Immunostaining of CD206 in RAW264.7 cells cultivated with MDA-MB-231con, MDA-MB-231miR-200c and MDA-MB-231miR-200c/siPAI-2 cells in the transwell system for 24 h. (B) CD206 positive cell area evaluated via immunostaining in MDA-MB-231con, MDA-MB-231miR-200c and MDA-MB-231miR-200c/siPAI-2 cells. (C) Schematic overview on the mechanisms by which miR-200c/PAI-2 axis regulates TAMs polarization and promotes triple-negative breast cancer metastasis.
used for comparison between groups, and a t-test was used for comparison between three independent samples. The value of P < 0.05 was considered as statistically significant. All experiments in this study were independently repeated for more than three times.
2.9. Transwell assay Transwell chambers (8 μm, BD Biosciences, San Diego, CA, USA) were used for co-culture assays. The bottom chamber was filled with 1 × 104 RAW264.7 cells containing 10% FBS culture medium. In all, 5 × 104 breast cancer cells MDA-MB-231con, MDA-MB-231miR-200c and MDA-MB-231miR-200c/siPAI-2 were suspended in serum-free medium and plated in the upper chamber, respectively. Establish a cell co-culture system. After culturing for 24 h in a 5% CO2, 37 °C incubator. The cells through the filter to the lower membrane surface were fixed in 1% paraformaldehyde for 1 min and stained with 0.1% crystal violet for 10 min. Cells were counted under a microscope at 200× magnification. Statistical results of migration cell numbers from three independent experiments were averaged from five image fields.
3. Results 3.1. miR-200 overexpression upregulates PAI-2 in MDA-MB-231 cell To further confirm the role of miR-200c and PAI-2 in breast cancer. We established MDA-MB-231miR-200ab/c cells that stably expressed miR200ab/c. Levels of PAI-2 were assessed by real-time RT-PCR in MDAMB-231 cells that overexpressed miR-200ab/c (MDA-MB-231miR-200ab/ c ) and MDA-MB-231 cells (MDA-MB-231con) (Fig. 1A). PAI-2 was upregulated in MDA-MB-231miR-200ab (P = 0.0004) and MDA-MB-231miR200c (P = 0.0001) cells relative to MDA-MB-231con (Fig. 1B). Immunofluorescence assays analysis showed that the number of PAI-2 green fluorescent particles increased significant and the fluorescence intensity was significantly enhanced in the cytoplasm of MDA-MB-231 cells treated with miR-200ab and miR-200c compared with the control group (Fig. 1C).
2.10. ELISA ELISA was conducted according to the manufacturer’s instructions. Concentrations of PAI-2 and IL-10 inthe culture supernatant of treated cells were measured with the use of a commercially available kit (CUSABIO). 2.11. Xenograft animal model
3.2. PAI-2 knockdown suppresses miR-200c overexpression-induced M2 phenotype polarization in lung metastatic mice
All animal experiments were approved and performed by the animal ethics committee of the Yanbian University, China. To investigate lung metastasis, 5 × 105 MDA-MB-231 cells transfected additionally with PAI-2 siRNA or scramble siRNA were suspended in 0.1 ml of PBS and injected into the tail veins of 6-week-old mice. Tumor-bearing mice were randomly assigned to one of three groups: MDA-MB-231 (n = 5); MDA-MB-231miR-200c (n = 5) and MDA-MB- 231miR-200c/siPAI-2 (n = 5).
miR-200c overexpression upregulated PAI-2 expression, and siRNAmediated PAI-2 knockdown successfully reduced PAI-2 levels in MDAMB-231miR-200c cells. Many studies have linked PAI-2 to TAMs in cancer [11]. But the exact mechanism in vivo is unclear. Therefore, in order to determine the in vivo dynamics of TAMs around TNBC cells after intravenous injection. At 56 days, strong bioluminescent signals were observed in the lungs of MDA-MB-231miR-200c (8 × 107 photon/sec/ cm2/sr) mice compared with MDA-MB-231con (5.4 × 105 photon/sec/ cm2/sr), but signals were decreased in MDA-MB-231miR-200c/siPAI-2 (1.5 × 107 photon/sec/cm2/sr) mice (Fig. 2A-B). H&E staining of lung sections showed that lung metastasis areas were greater in MDA-MB231miR-200c than in MDA-MB-231con (P < 0.05), and were reduced in
2.12. Statistical analysis The data analysis was performed using SPSS 17.0 software and GraphPad Prism7.0 software. The experimental data are expressed as the mean ± standard deviation. One-way analysis of variance was 5
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MDA-MB-231miR-200c/siPAI-2 (P < 0.05) (Fig. 2C-D). Lung metastatic tumor tissue immunohistochemistry (IHC) analyses revealed that miR200c overexpression upregulated F4/80 staining intensity in MDA-MB231 cells, whereas PAI-2 knockdown suppressed F4/80 staining intensity in MDA-MB-231miR-200c cells (Fig. 2 E-F). To investigate the effect of miR-200c on the polarization of tumor-associated macrophages, immunohistochemical analyses showed that miR-200c overexpression upregulated the expression of CD206 in MDA-MB-231 cells, whereas PAI-2 knockdown suppressed the expression of CD206 in MDAMB-231miR-200c cells (Fig. 2 G-H). This result suggested that miR-200c induces M2 phenotype polarization through the PAI-2 signaling pathway, which promotes the malignant progression of MDA-MB-231 cells.
CD206 in MDA-MB-231miR-200c cells compared to control group, whereas PAI-2 knockdown suppressed the staining intensity of CD206 in MDA-MB-231miR-200c cells (Fig. 4A-B). 4. Discussion Plasminogen activator system (uPA, PAI-2, SerpinE1) is characteristic of malignant tumors and is associated with its progression and metastasis. In recent years, the mechanisms of action of PAI-2 have been a hot topic in the field of oncology research. However, PAI-2 has been reported to be upregulated in cancer cells [13]. The role of miR200c (eg, migration, invasion, EMT) is similar to PAI-2 mediated properties. Studies have shown that PAI-2 mRNA levels are downregulated in cancerous sarcoma-derived Hs578TmiR-200c cells (MSL subtype) and ductal carcinoma-derived HCC-38miR-200c cells (BL1 subtype) [9]. According to our findings, PAI-2 mRNA levels in MDA-MB231miR-200c were upregulated. This indicates that PAI-2 is a new direct target of miR-200c. PAI-2 has been reported to play key roles in extracellular matrix remodeling, tumor growth, and metastasis in diverse cancers [14–16]. However, our results showed that miR-200c/PAI-2 axis had no significant effect on the proliferation ability of TNBC cells. In breast cancer patients and animal models, high miR-200c levels were associated with enhanced metastatic colonization and poor clinical outcomes [17,18]. Consistent with these observations, we found that the overexpression of miR-200c in MDA-MB-231 cells promoted lung metastasis in the mouse model. Our previous studies have shown that miR-200c can upregulate PAI-2 expression, but regarding the role of PAI-2 in lung metastasis, we demonstrated that miR-200c/PAI-2 axis inhibits the migration and pulmonary metastasis of MDA-MB-231 cells. The results of mouse xenotrans plantation showed that miR-200c overexpression and upregulation of PAI-2 may be the main mechanism of increased lung metastasis of MDA-MB-231 cells. Therefore, PAI-2 is the mediator of miR200c induced TNBC metastasis. A number of potential therapeutic strategies centered on macrophages are currently being studied [19]. Under the stimulation of breast cancer tumor microenvironment, TAM is close to M2 phenotype polarization, and M2-type TAM provides a favorable microenvironment for tumor growth [2]. Studies have shown that specific miRNAs play an important role in regulating TAM polarization and functional phenotype, and it is expected they may become a molecular target for tumor targeted diagnosis and treatment. Abnormally expressed miRNAs regulate the inflammatory phenotype of macrophages through alterin the morphological characteristics of macrophages [20]. Studies have shown that miRNAs clarify their effects by targeting multiple gene products, and miR-200c can enhance the polarization of TAM in vitro and in vivo [21]. Our studies have shown that miR-200c can upregulate PAI-2 expression, but whether the relationship between miR-200c and TAM is related to PAI-2 remains unclear. Regarding the role of PAI-2 in breast cancer, we demonstrated that miR-200c/PAI-2 axis induced F4/80 macrophage infiltration in MDA-MB-231 cells. Xenograft tissue sections showed that miR-200c overexpression or upregulation of PAI-2 is the main mechanisms for macrophage infiltration. Cancer cells communicate with neighboring cells via the miR-200c/ PAI-2 axis signaling pathway or other pathways to induce metastatic tumor growth, and macrophages exhibit plasticity through M1 to M2 transformation at different steps to enhance tumor metastasis and occurrence. The presence of M2-type macrophages is clinically associated with poor prognosis of various types of cancer [22], suggesting that macrophages induce metastatic development through unique cell interactions within metastatic sites. The increased number of M2-type macrophages was found in metastatic cancers and was considered an index to predict metastasis [23]. Many studies have shown a link between PAI-2 and M2-type TAM [24]. We observed CD206 infiltration in MDA-MB-231miR-200c xenograft tumors, while PAI-2 knockdown decreased this infiltration. In TNBC, IL-
3.3. RAW264.7 cells enhance miR-200c overexpression-induced migration in MDA-MB-231 cells In mouse xenograft tumor models, high miR-200c levels were associated with enhanced metastasis and colonization. To investigate the effect of PAI-2 on miR-200c-mediated migration of MDA-MB-231 cells, we performed transwell assays. In contrast, the migration ability was evidently increased in miR-200c overexpressed cells compared to control cells, while knockdown of PAI-2 significantly reduced the migration ability of MDA-MB-231miR-200c cells (Fig. 3 A-B). To investigate the effect of TAM on MDA-MB-231 cells migration mediated by miR-200c overexpression, RAW264.7 cells were co-cultured with MDA-MB231con, MDA-MB-231miR-200c and MDA-MB-231miR-200c/siPAI-2, respectively. Compared with the control group, miR-200c increased cell migration ability, while knockdown PAI-2 significantly reversed cell migration ability (Fig. 3 C-D). We evaluated how RAW264.7 cells promote miR-200c-mediated MDA-MB-231 migration in vitro. Through literature review, we learned that after co-culture with tumor cells and macrophages, the morphology of macrophages stretches, which is similar to that of macrophages in tumors. Therefore, we observed the morphology of RAW264.7 cells after 24 h of a co-culture system. Compared with the control group, miR-200c-mediated MDA-MB-231 cells significantly promoted the elongation of RAW264.7 cells, that exhibited a fusiform shape, while MDA-MB-231miR-200c/siPAI-2 cells maintained the original cell morphology of RAW264.7cells (Fig. 3E). Macrophages can secrete more anti-inflammatory cytokines and immunosuppressive factors, such as the M2-type macrophage induced differentiation factor IL-10. Therefore, after co-culture of RAW264.7 cells with MDA-MB-231con, MDA-MB-231miR-200c and MDA-MB-231miR200c/siPAI-2 , respectively. ELISA assay showed that knocking down PAI-2 could reverse the secretion of IL-10 promoted by the overexpression of miR-200c (Fig. 3F). This suggests that knockdown of PAI-2 blocks miR200c-induced M2 phenotype polarization. In order to exclude the influence of other factors of tumor secretion on RAW264.7, we evaluated the changes of PAI-2 expression in the culture medium of RAW264.7 cells and MDA-MB-231con, MDA-MB-231miR-200c and MDA-MB-231miR200c/siPAI-2 cells in the co-culture system. Compared with the control group, miR-200c-mediated MDA-MB-231 cells significantly increased the secretion of PAI-2, while decreased the secretion of PAI-2 in MDAMB-231miR-200c/siPAI-2 cells (Fig. 3G). This suggested that the morphological and phenotypic changes of RAW264.7 cells were mediated by PAI-2 and not by other factors. 3.4. miR-200c/PAI-2 axis promotes the malignant progression of MDA-MB231 cells by inducing M2 phenotype polarization Previous results showed that miR-200c/PAI-2 axis signaling pathway could promote the polarization of macrophages in vivo. In order to evaluate the effect of miR-200c/PAI-2 axis signaling pathway on RAW264.7 cells M2 phenotype polarization in vitro. We investigated the expression of CD206. In transwell co-culture of RAW264.7 cells and MDA-MB-231 cells, we demonstrated increased staining intensity of 6
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References
10 is mainly secreted by cancer cells, and polarizes TAMs to the M2 phenotype, which in turn promotes cancer cell migration and metastasis [25]. In the present study, we investigated the role of miR-200c/ PAI-2 axis in cytokines production in human TNBC cells. Studies on the co-culture of MDA-MB-231 and RAW264.7 cells to simulate in vivo microenvironment. We found that IL-10 was elevated in miR-200c overexpressed cancer cells but declined in knockdown PAI-2 cells. Therefore, we concluded that miR-200c/PAI-2 axis increases the expression and secretion of IL-10 in TNBC cells and eventually promotes M2 macrophage polarization. The results showed that PAI-2 promotes the malignant progression of TNBC cells through M2 phenotype polarization. In this study, we first investigate the relationship of miR200c, PAI-2 and TAM in TNBC tumor tissues. The present study reports a new biological role for miR-200c/PAI-2 axis in promoting the alternative activation of TAMs and promoting breast tumor metastasis. miR-200c/PAI-2 axis increased the polarization of TAMs towards the M2 phenotype by promoting IL-10 expression in TNBC cells. Overall, we uncovered a novel role for miR-200c in the regulation of macrophage polarization and tumor metastasis in TNBC (Fig. 4C). These findings suggest that targeting the miR-200c/PAI-2 axis signaling pathway may be a potential therapeutic strategy for the treatment of TNBC.
[1] Q.X. Wang, Y.F. Cai, Y.Y. Chen, et al., Additional prognostic value of lymph node ratio (LNR) and number of negative lymph nodes (NLNs) in Chinese patients with triple negative breast cancer, Ann. Clin. Lab. Sci. 47 (1) (2017) 68–75. [2] J. Choi, J. Gyamfi, H. Jang, et al., The role of tumor-associated macrophage in breast cancer biology, Histol. Histopathol. 33 (2) (2018) 133–145. [3] S. Singh, N. Mehta, J. Lilan, et al., Initiative action of tumor-associated macrophage during tumor metastasis, Biochim. Open 4 (2017) 8–18. [4] M. Orecchioni, Y. Ghosheh, A.B. Pramod, K. Ley, Macrophage polarization: different gene signatures in M1(LPS+) vs. classically and M2(LPS-) vs. alternatively activated macrophages, Front. Immunol. 10 (2019) 1084. [5] A. Mantovani, F. Marchesi, A. Malesci, L. Laghi, P. Allavena, Tumour-associated macrophages as treatment targets in oncology, Nat. Rev. Clin. Oncol. 14 (7) (2017) 399–416. [6] Y.J. Piao, H.S. Kim, E.H. Hwang, J. Woo, M. Zhang, W.K. Moon, Breast cancer cellderived exosomes and macrophage polarization are associated with lymph node metastasis, Oncotarget. 9 (7) (2017) 7398–7410. [7] I.S. Pateras, T. Cooks, Determination of polarization of resident macrophages and their effect on the tumor microenvironment, Methods MolBiol. 1928 (2019) 101–112. [8] J. Ming, Y. Zhou, J. Du, et al., miR-381 suppresses C/EBPα-dependent Cx43 expression in breast cancer cells, Biosci. Rep. (2015) 35(6). [9] T. Jin, H.S. Kim, S.K. Choi, et al., microRNA-200c/141 upregulates SerpinB2 to promote breast cancer cell metastasis and reduce patient survival, Oncotarget. 8 (20) (2017) 32769–32782. [10] N. Li, Jun-Fang Qin, X. Han, et al., miR-21a negatively modulates tumor suppressor genes PTEN and miR-200c and further promotes the transformation of M2 macrophages, Immunol. Cell Biol. (2018) 96. [11] K. Ohba, Y. Miyata, S. Kanda, et al., Expression of urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor and plasminogen activator inhibitors in patients with renal cell carcinoma: correlationwith tumor associated macrophage and prognosis, J. Urology 174 (2) (2005) 461–465. [13] W.A. Schroder, L. Major, A. Suhrbier, The role of SerpinB2 in immunity, Crit. Rev. Immunol. 31 (1) (2011) 15–30. [14] D.R. Croucher, D.N. Saunders, S. Lobov, et al., Revisiting the biological roles of PAI2 (SERPINB2) in cancer, Nat. Rev. Cancer 8 (7) (2008) 535–545. [15] M. Valiente, A.C. Obenauf, X. Jin, Q. Chen, X.H. Zhang, D.J. Lee, J.E. Chaft, M.G. Kris, J.T. Huse, E. Brogi, J. Massagué, Serpins promote cancer cell survival and vascular co-option in brain metastasis, Cell 156 (2014) 1002–1016. [16] P. Utaijaratrasmi, K. Vaeteewoottacharn, T. Tsunematsu, et al., The microRNA-15aPAI-2 axis in cholangiocarcinoma-associated fibroblasts promotes migration of cancer cells, Mol. Cancer 17 (1) (2018) 10. [17] M. Tuomarila, K. Luostari, Y. Soini, et al., Overexpression of MicroRNA-200c predicts poor outcome in patients with PR-negative breast cancer, PLoS ONE 9 (9) (2014) e109508. [18] M.T.N. Le, P. Hamar, C. Guo, et al., miR-200–containing extracellular vesicles promote breast cancer cell metastasis, J. Clin. Invest. 124 (12) (2014) 5109–5128. [19] A. Mantovani, F. Marchesi, A. Malesci, et al., Tumour-associated macrophages as treatment targets in oncology, Nat. Rev. Clin. Oncol. 14 (7) (2017) 399–416. [20] K. Essandoh, Y. Li, J. Huo, G.C. Fan, MiRNA-mediated macrophage polarization and its potential role in the regulation of inflammatory response, Shock 46 (2) (2016) 122–131. [21] M. Tran, S.M. Lee, D.J. Shin, et al., Loss of miR-141/200c ameliorates hepatic steatosis and inflammation by reprogramming multiple signaling pathways in NASH, Jci Insight (2017) 2(21). [22] S.K. Biswas, A. Sica, C.E. Lewis, Plasticity of macrophage function during tumor progression: regulation by distinct molecular mechanisms, J. Immunol. 180 (2008) 2011–2017. [23] B. Zhang, M. Cao, Y. He, et al., Increased circulating M2-like monocytes in patients with breast cancer, Tumor Biol. (2017) 39(6). [24] W.A. Schroder, T.D. Hirata, T.T. Le, et al., SerpinB2 inhibits migration and promotes a resolution phase signature in large peritoneal macrophages, Sci. Rep. (2019) 9(1). [25] N. Wang, W. Liu, Y. Zheng, et al., CXCL1 derived from tumor-associated macrophages promotes breast cancer metastasis via activating NF-κB/SOX4 signaling, Cell Death Dis. 9 (9) (2018) 880.
Compliance with ethical standards Funding This research was supported by the National Natural Science Funds of China (Nos. 81560478 and 81660394), Key Laboratory of Natural Resources of Changbai Mountain, Project of Science and Technology Department of Jilin Province (No. 20180414087GH). Ethical approval “All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.” “This article does not contain any studies with human participants performed by any of the authors.” Declaration of Competing Interest The authors declare that they have no conflict of interest. Acknowledgements This study was funded the National Natural Science Funds of China (grant nos. 81560478 and 81660394), Key Laboratory of Natural Resources of Changbai Mountain, Project of Science and Technology Department of Jilin Province (grant no. 20180414087GH). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.intimp.2019.106028.
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
miR-200c/PAI-2 promotes the progression of triple negative breast cancer via M1/M2 polarization induction of macrophage ⁎
⁎
Ziqi Menga, Rui Zhanga, Yixuan Wanga, Guang Zhua, Tiefeng Jina, , Chunguo Lic, , ⁎ Songnan Zhangb, a
Department of Pathology and Cancer Research Center, Yanbian University Medical College, Yanji 133002, China Department of Oncology, Yanbian University Hospital, Yanji 133002, China c Department of Oncology, Jilin City Central Hospital, Jilin 132011, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: miR-200c PAI-2 MDA-MB-231 Macrophages Cell migration Transwell co-culture
Purpose: To investigate the effect of miR-200c/PAI-2 on macrophage polarization into M2-type TAMs in TNBC. Methods and materials: PAI-2 expression in MDA-MB-231con, MDA-MB-231miR-200ab and MDA-MB-231miR-200c breast cancer cells was evaluated by RT-PCR and immunofluorescence (IF), while the expression of the TAM marker F4/80 and the M2-type TAM marker CD206 in MDA-MB-231con, MDA-MB-231miR-200c and MDA-MB231miR-200c/siPAI-2 mouse lung metastatic tumor tissues was examined with immunohistochemistry (IHC). The effects of RAW264.7 cells on MDA-MB-231con, MDA-MB-231miR-200c and MDA-MB-231miR-200c/siPAI-2 were examined by transwell co-culture. CD206 expression in RAW264.7 cells were confirmed by immunostaining. The level of PAI-2 and IL-10 in the co-culture supernatants were assessed using ELISA. Results: 1. RT-PCR and IF analysis showed that PAI-2 was upregulated in MDA-MB-231miR-200c cells. 2. IHC assays analysis showed that the numbers of F4/80 and CD206 positive cells were increased in MDA-MB-231miR200c tumor tissues, while in MDA-MB-231miR-200c/siPAI-2 tumor tissues were decreased. 3. Transwell co-culture assays analysis showed that MDA-MB-231miR-200c cells significantly promoted the cell migration ability compared with the control group, while knockdown PAI-2 significantly inhibited the cell migration ability (P < 0.05). 4. Transwell co-culture and immunostaining assays analysis showed that overexpression miR-200c in MDA-MB-231 cell line increased the CD206 expression in RAW264.7 cells, while knockdown PAI-2 decreased. 5. ELISA assays analysis showed that miR-200c-mediated MDA-MB-231 cells significantly increased the secretion of PAI-2 and IL-10, while decreased the secretion of PAI-2 and IL-10 in MDA-MB-231 miR-200c/siPAI-2 cells. Conclusions: miR-200c promotes the malignant progressions of TNBC by PAI-2 upregulation and M2 phenotype macrophages polarization.
1. Introduction
important components of the tumor microenvironment, accounting for 50%-80% of interstitial cells. Studies have shown that the numbers of invasive TAMs are positively correlated with breast cancer staging, grading, metastatic potential, and prognosis [2]. Though macrophages are an important part of the body's innate immunity and play a role in tumor immunity during tumor initiation [3], macrophages are highly plastic. Under the inductions of certainfactors, the phenotypes, functions and morphologies of macrophages will be changed and finally lead to macrophage polarization [4]. TAMs can be classified into a classical pathway-activated macrophages (M1-type) and alternative pathway-activated macrophages (M2-type). M1-type TAMs mainly shows an anti-cancer effect, while M2-type TAMs mainly shows the
Breast cancer can be divided into four subtypes, luminal A, luminal B, HER2 and triple-negative breast cancer (TNBC). The biological behavior and treatment strategies of these different molecular subtypes are completely different. Patients with TNBC, due to the lack of specific molecular targets, they usually faced many problems such as early recurrence, rapid malignant progressions and a short overall survival time, the five-year overall survival rate for patients with TNBC is only 64.97% [1]. Thus, it is important to find new treatment strategies. The tumor microenvironment is composed of tumor cells, mesenchymal cells and immune cells. Tumor-associated macrophages are
⁎ Corresponding authors at: Yanbian University, 977 Park Road, Yanji City, Jilin Province, China (T. Jin). Jilin City Central Hospital, Jilin City, Jilin Province, China (C. Li). Yanbian Hospital, Yanji City, Jilin Province, China (S. Zhang). E-mail addresses: [email protected] (T. Jin), [email protected] (C. Li), [email protected] (S. Zhang).
https://doi.org/10.1016/j.intimp.2019.106028 Received 12 August 2019; Received in revised form 21 October 2019; Accepted 4 November 2019 1567-5769/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Ziqi Meng, et al., International Immunopharmacology, https://doi.org/10.1016/j.intimp.2019.106028
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Fig. 1. miR-200 overexpression upregulated PAI-2 in MDA-MB-231 cells. (A) Representative images and (B) complete data of PAI-2 RT-PCR of MDA-MB-231con and MDA-MB-231miR-200ab/c cells. (C) Immunofluorescence staining of PAI-2 in MDA-MB-231miR-200ab/c cells and MDA-MB-231con cells (×400).
2. Materials and methods
opposite [5]. Recently, many studies discovered that TAMs in tumor tissues tend to polarize into the M2 phenotype [6], and this effect mainly depends on cancer types and stromal interactions. Macrophage polarization is confirmed as a key process in cancer progression and macrophages express different states in response to tumor microenvironmental signals [7]. Choi J. et al. confirmed that M2-type TAMs promote tumor invasion, metastasis, immunosuppression and drug resistance by inducing angiogenesis, degrading the tumor extracellular matrix and helping tumor cells escape from immune regulation during the progression of breast cancer [2]. Therefore, reversing the polarization of M2-type TAMs, reducing their recruitment and blocking the tumor-promoting function of M2-type TAMs could be a new anti-tumor strategy. The miR-200 family (including miR-200a, miR-200b, miR-200c, miR-141 and miR-429) is an important group of miRNAs. It plays an important role in maintaining epithelial traits in cells or tissues and in regulates tumor proliferation, invasion and metastasis. Studies have found that the expression level of miR-200 is closely related to the prognosis of breast cancer patients [8], but there are few reports on whether it influences the TAMs polarization. This study explored the effects of miR-200 on the polarization of TAMs and the biological characteristics of TNBC, and it provides new ideas to further understand the role of miR-200 in TAMs polarization and the development of TNBC. Our previous study showed that overexpression miR-200 upregulated the expression of plasminogen activator inhibitor-2 (PAI-2) in MDA-MB-231 cells [9]. The expression of miR-200c in breast cancer cells can promote the polarization of M2-type macrophages [10]. However, the exact role of PAI-2 in TAM polarization and its underlying mechanisms remain unknown. This study aimed to clarify whether PAI-2 is involved in miR-200c-mediated M2 phenotypic polarization.
2.1. Cell culture Mouse macrophage cells RAW264.7 and human breast cancer cells MDA-MB-231 were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cell lines were cultured in RPMI1640 and DMEM medium respectively, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C, 5% CO2.
2.2. Immunohistochemistry The tissue was fixed with 10% formalin and embedded in paraffin blocks. Paraffin-embedded continuous tissue sections were used for immunohistochemical staining of F4/80 and CD206. Briefly, 4 µm tissue sections were subsequently dewaxed and rehydrated using xylene and gradient alcohol washes. Antigen retrieval was performed at 121 °C for 1 min using pH 6.0 citrate buffer. After hydrogen peroxide blocking at room temperature for 20 min, sections were incubated with primary polyclonal antibodies against F4/80 (Thermo Fisher Scientific Inc.) and CD206 (Cell Signaling Technology, 91992, USA) overnight at 4 °C. After incubation with the secondary antibody at room temperaturefor 1 h, immunostaining with DAB and counterstaining of the slide with hematoxylin, TAMs were scored as the infiltration density of F4/80+ or CD206+ cells inbrown-yellow monocyte/macrophage morphology. TAMs were evaluated by adapting the reported hotspot quantitative method. The entire stained section was first scanned at low magnification (×200) to identify the hotspot areas (five areas of tumor stroma) with the most abundant macrophages. The average count of macrophages in all five areas and the density of infiltration was estimated (per 0.24 mm2) at higher magnification (×400). Two researchers (pathologists) blinded to the information carried out the evaluation process. 2
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Fig. 2. PAI-2 knockdown decreased M2 phenotype polarization by miR-200c overexpression in MDA-MB-231 cells. (A) Representative BLI of mice injected with MDA-MB-231con, MDA-MB-231miR-200c and MDA-MB-231miR-200c/siPAI-2 cells. (B) Total photon flux of the bioluminescent signals emitted from thorax regions of MDAMB-231con, MDA-MB-231miR-200c and MDA-MB-231miR-200c/siPAI-2 mice. (C) Representative H&E staining in lung tissues. (D) Tumor area of individual lung metastases evaluated via H&E staining in MDA-MB-231con, MDA-MB-231miR-200c and MDA-MB-231miR-200c/siPAI-2 mice. (E) Representative F4/80 immunostaining in lung tissues. (F) F4/80 tumor staining area of individual lung metastases evaluated via IHC staining. (G) Representative CD206 immunostaining in lung tissues. (H) Randomly chosen staining area of CD206 positive macrophages in lung metastatic tumor around in the 5 field of view (×200).
and visualized by ethidium bromide. All assays were performed at least three times.
2.3. Real-time PCR Total RNA was isolated by use of TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed using a Prime Script™ RTPCR Kit (Takara, RR014A). Real-time PCRs reactions were run on an ABI PRISM 7900 utilizing a SYBR Green PCR master mix (Applied Biosystems, Foster City, CA, USA) and specific primer sets for PAI-2. After amplification, PCR products were separated on 1% agarose gels
2.4. Colony formation assay Cells were plated on 6-well plates at a concentration of 5 × 103 cells for each well and incubated for 12 days at 37 °C. The cells were fixed in 4% paraformaldehyde for 15 min and then stained with Giemsa for 3
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Fig. 3. RAW264.7 cells promote the migration of MDA-MB-231 cells. (A-B) Transwell migration assay of MDA-MB231con, MDA-MB-231miR-200c and MDAMB-231miR-200c/siPAI-2 for 24 h. (C-D) Transwell migration assay of 1 × 104 RAW264.7 cells that were co-cultured with MDA-MB-231con, MDA-MB231miR-200c and MDA-MB-231miR-200c/ siPAI-2 for 24 h. (E) Cell morphology image of RAW264.7 cells co-cultured MDA-MBwith MDA-MB-231con, miR-200c and MDA-MB-231miR-200c/ 231 siPAI-2 for 24 h. (F) The level of IL-10 in the co-culture supernatants was assessed using ELISA. (G) The level of PAI-2 in the co-culture supernatants was assessed using ELISA (All values from three independent experiments are quantified as the mean ± SD, P < 0.05).
2.7. Lentiviral transduction
25 min after being washed with cold PBS.
The pLent-H1-GFP-Puro-based lentiviruses carrying luciferase were purchased from Vigene Biosciences and transfected into MDA-MB-231 cells using DMEM medium containing polybrene (5 μg/ml), respectively. Lentiviral vectors containing the miR-200ab/c constructs were kindly supplied by Dr. Gregory J. Goodall. After transduction of miR200c lentivirus into MDA-MB-231 cells and selection with puromycin (3 μg/ml), MDA-MB-231 cells overexpressing miR-200ab/c were sorted using a FACS caliber flow cytometer (BD Biosciences, CA, USA). Additionally, lentivirus was transduced into miR-200ab/c-overexpressing MDA-MB-231 cells for animal studies. MDA-MB-231 cells and miR-200ab/c-overexpressing MDA-MB-231 cells are referred to as MDA-MB-231con and MDA-MB-231miR-200ab/c.
2.5. MTT assay Cells were seeded onto 96-well plates at a density of 5 × 103 cells per well and incubated for 0, 24, 48, 72 h. 100 μL medium containing 20 μL of MTT reagent (5 mg/mL) was added into each well and incubated for 4 h under the same conditions. Then, 100 μL of DMSO was added to each well and the absorbance value (OD) at 490 nm of each well was measured using a microplate reader (LabSystemsMultiskan Ascent). Five wells per group at least were analyzed and repeated three times.
2.6. Transfection 2.8. Immunofluorescence We purchased three different PAI-2 siRNA from Bioneer. siRNA sequences were as follows: (1) PAI-2 siRNA sense, AAGGUAUCCCUA UUUUUGAAGCCUGUCUC and antisense, AACUUCAAAAAUAGGGAU ACCCCUGUCUC (2) PAI-2 siRNA sense, GAGCUUCCGGGAAGAAUAU and antisense, AUAUUCUUCCCGGAAGCUC (3) PAI-2 siRNA sense, CAGAGAACAACCAGAUUGA and antisense, UCAAUCUGGUUGUUCU CUG. Scramble siRNA (AccuTarget™ Negative Control siRNA, BioRP) was also used. Cells were transfected with 30 nM PAI-2 siRNA or scramble siRNA using Lipofectamine-3000 (Invitrogen).
Cells grown in 6-well plates culture slides were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.5% Triton X-100 (CWBIO, China) and blocked with 3% BSA for 2 h. Cells were incubated with primary antibody in 3% BSA at 4 °C overnight, washed three times with PBS, incubated with Alexa Fluor 568-labeled secondary antibody (Invitrogen) in 3% BSA for 2 h, and then analyzed by Leica SP5II confocal microscope.
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Fig. 4. miR-200c/PAI-2 axis promotes the malignant progression of triple- negative breast cancer cells by inducing the polarization of macrophages into M2 direction. (A) Immunostaining of CD206 in RAW264.7 cells cultivated with MDA-MB-231con, MDA-MB-231miR-200c and MDA-MB-231miR-200c/siPAI-2 cells in the transwell system for 24 h. (B) CD206 positive cell area evaluated via immunostaining in MDA-MB-231con, MDA-MB-231miR-200c and MDA-MB-231miR-200c/siPAI-2 cells. (C) Schematic overview on the mechanisms by which miR-200c/PAI-2 axis regulates TAMs polarization and promotes triple-negative breast cancer metastasis.
used for comparison between groups, and a t-test was used for comparison between three independent samples. The value of P < 0.05 was considered as statistically significant. All experiments in this study were independently repeated for more than three times.
2.9. Transwell assay Transwell chambers (8 μm, BD Biosciences, San Diego, CA, USA) were used for co-culture assays. The bottom chamber was filled with 1 × 104 RAW264.7 cells containing 10% FBS culture medium. In all, 5 × 104 breast cancer cells MDA-MB-231con, MDA-MB-231miR-200c and MDA-MB-231miR-200c/siPAI-2 were suspended in serum-free medium and plated in the upper chamber, respectively. Establish a cell co-culture system. After culturing for 24 h in a 5% CO2, 37 °C incubator. The cells through the filter to the lower membrane surface were fixed in 1% paraformaldehyde for 1 min and stained with 0.1% crystal violet for 10 min. Cells were counted under a microscope at 200× magnification. Statistical results of migration cell numbers from three independent experiments were averaged from five image fields.
3. Results 3.1. miR-200 overexpression upregulates PAI-2 in MDA-MB-231 cell To further confirm the role of miR-200c and PAI-2 in breast cancer. We established MDA-MB-231miR-200ab/c cells that stably expressed miR200ab/c. Levels of PAI-2 were assessed by real-time RT-PCR in MDAMB-231 cells that overexpressed miR-200ab/c (MDA-MB-231miR-200ab/ c ) and MDA-MB-231 cells (MDA-MB-231con) (Fig. 1A). PAI-2 was upregulated in MDA-MB-231miR-200ab (P = 0.0004) and MDA-MB-231miR200c (P = 0.0001) cells relative to MDA-MB-231con (Fig. 1B). Immunofluorescence assays analysis showed that the number of PAI-2 green fluorescent particles increased significant and the fluorescence intensity was significantly enhanced in the cytoplasm of MDA-MB-231 cells treated with miR-200ab and miR-200c compared with the control group (Fig. 1C).
2.10. ELISA ELISA was conducted according to the manufacturer’s instructions. Concentrations of PAI-2 and IL-10 inthe culture supernatant of treated cells were measured with the use of a commercially available kit (CUSABIO). 2.11. Xenograft animal model
3.2. PAI-2 knockdown suppresses miR-200c overexpression-induced M2 phenotype polarization in lung metastatic mice
All animal experiments were approved and performed by the animal ethics committee of the Yanbian University, China. To investigate lung metastasis, 5 × 105 MDA-MB-231 cells transfected additionally with PAI-2 siRNA or scramble siRNA were suspended in 0.1 ml of PBS and injected into the tail veins of 6-week-old mice. Tumor-bearing mice were randomly assigned to one of three groups: MDA-MB-231 (n = 5); MDA-MB-231miR-200c (n = 5) and MDA-MB- 231miR-200c/siPAI-2 (n = 5).
miR-200c overexpression upregulated PAI-2 expression, and siRNAmediated PAI-2 knockdown successfully reduced PAI-2 levels in MDAMB-231miR-200c cells. Many studies have linked PAI-2 to TAMs in cancer [11]. But the exact mechanism in vivo is unclear. Therefore, in order to determine the in vivo dynamics of TAMs around TNBC cells after intravenous injection. At 56 days, strong bioluminescent signals were observed in the lungs of MDA-MB-231miR-200c (8 × 107 photon/sec/ cm2/sr) mice compared with MDA-MB-231con (5.4 × 105 photon/sec/ cm2/sr), but signals were decreased in MDA-MB-231miR-200c/siPAI-2 (1.5 × 107 photon/sec/cm2/sr) mice (Fig. 2A-B). H&E staining of lung sections showed that lung metastasis areas were greater in MDA-MB231miR-200c than in MDA-MB-231con (P < 0.05), and were reduced in
2.12. Statistical analysis The data analysis was performed using SPSS 17.0 software and GraphPad Prism7.0 software. The experimental data are expressed as the mean ± standard deviation. One-way analysis of variance was 5
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MDA-MB-231miR-200c/siPAI-2 (P < 0.05) (Fig. 2C-D). Lung metastatic tumor tissue immunohistochemistry (IHC) analyses revealed that miR200c overexpression upregulated F4/80 staining intensity in MDA-MB231 cells, whereas PAI-2 knockdown suppressed F4/80 staining intensity in MDA-MB-231miR-200c cells (Fig. 2 E-F). To investigate the effect of miR-200c on the polarization of tumor-associated macrophages, immunohistochemical analyses showed that miR-200c overexpression upregulated the expression of CD206 in MDA-MB-231 cells, whereas PAI-2 knockdown suppressed the expression of CD206 in MDAMB-231miR-200c cells (Fig. 2 G-H). This result suggested that miR-200c induces M2 phenotype polarization through the PAI-2 signaling pathway, which promotes the malignant progression of MDA-MB-231 cells.
CD206 in MDA-MB-231miR-200c cells compared to control group, whereas PAI-2 knockdown suppressed the staining intensity of CD206 in MDA-MB-231miR-200c cells (Fig. 4A-B). 4. Discussion Plasminogen activator system (uPA, PAI-2, SerpinE1) is characteristic of malignant tumors and is associated with its progression and metastasis. In recent years, the mechanisms of action of PAI-2 have been a hot topic in the field of oncology research. However, PAI-2 has been reported to be upregulated in cancer cells [13]. The role of miR200c (eg, migration, invasion, EMT) is similar to PAI-2 mediated properties. Studies have shown that PAI-2 mRNA levels are downregulated in cancerous sarcoma-derived Hs578TmiR-200c cells (MSL subtype) and ductal carcinoma-derived HCC-38miR-200c cells (BL1 subtype) [9]. According to our findings, PAI-2 mRNA levels in MDA-MB231miR-200c were upregulated. This indicates that PAI-2 is a new direct target of miR-200c. PAI-2 has been reported to play key roles in extracellular matrix remodeling, tumor growth, and metastasis in diverse cancers [14–16]. However, our results showed that miR-200c/PAI-2 axis had no significant effect on the proliferation ability of TNBC cells. In breast cancer patients and animal models, high miR-200c levels were associated with enhanced metastatic colonization and poor clinical outcomes [17,18]. Consistent with these observations, we found that the overexpression of miR-200c in MDA-MB-231 cells promoted lung metastasis in the mouse model. Our previous studies have shown that miR-200c can upregulate PAI-2 expression, but regarding the role of PAI-2 in lung metastasis, we demonstrated that miR-200c/PAI-2 axis inhibits the migration and pulmonary metastasis of MDA-MB-231 cells. The results of mouse xenotrans plantation showed that miR-200c overexpression and upregulation of PAI-2 may be the main mechanism of increased lung metastasis of MDA-MB-231 cells. Therefore, PAI-2 is the mediator of miR200c induced TNBC metastasis. A number of potential therapeutic strategies centered on macrophages are currently being studied [19]. Under the stimulation of breast cancer tumor microenvironment, TAM is close to M2 phenotype polarization, and M2-type TAM provides a favorable microenvironment for tumor growth [2]. Studies have shown that specific miRNAs play an important role in regulating TAM polarization and functional phenotype, and it is expected they may become a molecular target for tumor targeted diagnosis and treatment. Abnormally expressed miRNAs regulate the inflammatory phenotype of macrophages through alterin the morphological characteristics of macrophages [20]. Studies have shown that miRNAs clarify their effects by targeting multiple gene products, and miR-200c can enhance the polarization of TAM in vitro and in vivo [21]. Our studies have shown that miR-200c can upregulate PAI-2 expression, but whether the relationship between miR-200c and TAM is related to PAI-2 remains unclear. Regarding the role of PAI-2 in breast cancer, we demonstrated that miR-200c/PAI-2 axis induced F4/80 macrophage infiltration in MDA-MB-231 cells. Xenograft tissue sections showed that miR-200c overexpression or upregulation of PAI-2 is the main mechanisms for macrophage infiltration. Cancer cells communicate with neighboring cells via the miR-200c/ PAI-2 axis signaling pathway or other pathways to induce metastatic tumor growth, and macrophages exhibit plasticity through M1 to M2 transformation at different steps to enhance tumor metastasis and occurrence. The presence of M2-type macrophages is clinically associated with poor prognosis of various types of cancer [22], suggesting that macrophages induce metastatic development through unique cell interactions within metastatic sites. The increased number of M2-type macrophages was found in metastatic cancers and was considered an index to predict metastasis [23]. Many studies have shown a link between PAI-2 and M2-type TAM [24]. We observed CD206 infiltration in MDA-MB-231miR-200c xenograft tumors, while PAI-2 knockdown decreased this infiltration. In TNBC, IL-
3.3. RAW264.7 cells enhance miR-200c overexpression-induced migration in MDA-MB-231 cells In mouse xenograft tumor models, high miR-200c levels were associated with enhanced metastasis and colonization. To investigate the effect of PAI-2 on miR-200c-mediated migration of MDA-MB-231 cells, we performed transwell assays. In contrast, the migration ability was evidently increased in miR-200c overexpressed cells compared to control cells, while knockdown of PAI-2 significantly reduced the migration ability of MDA-MB-231miR-200c cells (Fig. 3 A-B). To investigate the effect of TAM on MDA-MB-231 cells migration mediated by miR-200c overexpression, RAW264.7 cells were co-cultured with MDA-MB231con, MDA-MB-231miR-200c and MDA-MB-231miR-200c/siPAI-2, respectively. Compared with the control group, miR-200c increased cell migration ability, while knockdown PAI-2 significantly reversed cell migration ability (Fig. 3 C-D). We evaluated how RAW264.7 cells promote miR-200c-mediated MDA-MB-231 migration in vitro. Through literature review, we learned that after co-culture with tumor cells and macrophages, the morphology of macrophages stretches, which is similar to that of macrophages in tumors. Therefore, we observed the morphology of RAW264.7 cells after 24 h of a co-culture system. Compared with the control group, miR-200c-mediated MDA-MB-231 cells significantly promoted the elongation of RAW264.7 cells, that exhibited a fusiform shape, while MDA-MB-231miR-200c/siPAI-2 cells maintained the original cell morphology of RAW264.7cells (Fig. 3E). Macrophages can secrete more anti-inflammatory cytokines and immunosuppressive factors, such as the M2-type macrophage induced differentiation factor IL-10. Therefore, after co-culture of RAW264.7 cells with MDA-MB-231con, MDA-MB-231miR-200c and MDA-MB-231miR200c/siPAI-2 , respectively. ELISA assay showed that knocking down PAI-2 could reverse the secretion of IL-10 promoted by the overexpression of miR-200c (Fig. 3F). This suggests that knockdown of PAI-2 blocks miR200c-induced M2 phenotype polarization. In order to exclude the influence of other factors of tumor secretion on RAW264.7, we evaluated the changes of PAI-2 expression in the culture medium of RAW264.7 cells and MDA-MB-231con, MDA-MB-231miR-200c and MDA-MB-231miR200c/siPAI-2 cells in the co-culture system. Compared with the control group, miR-200c-mediated MDA-MB-231 cells significantly increased the secretion of PAI-2, while decreased the secretion of PAI-2 in MDAMB-231miR-200c/siPAI-2 cells (Fig. 3G). This suggested that the morphological and phenotypic changes of RAW264.7 cells were mediated by PAI-2 and not by other factors. 3.4. miR-200c/PAI-2 axis promotes the malignant progression of MDA-MB231 cells by inducing M2 phenotype polarization Previous results showed that miR-200c/PAI-2 axis signaling pathway could promote the polarization of macrophages in vivo. In order to evaluate the effect of miR-200c/PAI-2 axis signaling pathway on RAW264.7 cells M2 phenotype polarization in vitro. We investigated the expression of CD206. In transwell co-culture of RAW264.7 cells and MDA-MB-231 cells, we demonstrated increased staining intensity of 6
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References
10 is mainly secreted by cancer cells, and polarizes TAMs to the M2 phenotype, which in turn promotes cancer cell migration and metastasis [25]. In the present study, we investigated the role of miR-200c/ PAI-2 axis in cytokines production in human TNBC cells. Studies on the co-culture of MDA-MB-231 and RAW264.7 cells to simulate in vivo microenvironment. We found that IL-10 was elevated in miR-200c overexpressed cancer cells but declined in knockdown PAI-2 cells. Therefore, we concluded that miR-200c/PAI-2 axis increases the expression and secretion of IL-10 in TNBC cells and eventually promotes M2 macrophage polarization. The results showed that PAI-2 promotes the malignant progression of TNBC cells through M2 phenotype polarization. In this study, we first investigate the relationship of miR200c, PAI-2 and TAM in TNBC tumor tissues. The present study reports a new biological role for miR-200c/PAI-2 axis in promoting the alternative activation of TAMs and promoting breast tumor metastasis. miR-200c/PAI-2 axis increased the polarization of TAMs towards the M2 phenotype by promoting IL-10 expression in TNBC cells. Overall, we uncovered a novel role for miR-200c in the regulation of macrophage polarization and tumor metastasis in TNBC (Fig. 4C). These findings suggest that targeting the miR-200c/PAI-2 axis signaling pathway may be a potential therapeutic strategy for the treatment of TNBC.
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Compliance with ethical standards Funding This research was supported by the National Natural Science Funds of China (Nos. 81560478 and 81660394), Key Laboratory of Natural Resources of Changbai Mountain, Project of Science and Technology Department of Jilin Province (No. 20180414087GH). Ethical approval “All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.” “This article does not contain any studies with human participants performed by any of the authors.” Declaration of Competing Interest The authors declare that they have no conflict of interest. Acknowledgements This study was funded the National Natural Science Funds of China (grant nos. 81560478 and 81660394), Key Laboratory of Natural Resources of Changbai Mountain, Project of Science and Technology Department of Jilin Province (grant no. 20180414087GH). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.intimp.2019.106028.
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