miRNA let-7b modulates macrophage polarization and enhances tumor-associated macrophages to promote angiogenesis and mobility in prostate cancer

Macrophage polarization is a highly plastic physiological process that responds to a variety of environmental factors by changing macrophage phenotype and function. Tumor-associated macrophages (TAMs) are generally recognized as promoting tumor progression. As universal regulators, microRNAs (miRNAs) are functionally involved in numerous critical cellular processes including macrophage polarization. Let-7b, a miRNA, has differential expression patterns in inflamed tissues compared with healthy controls. However, whether and how miRNA let-7b regulates macrophage phenotype and function is unclear. In this report, we find that up-regulation of let-7b is characteristic of prostatic TAMs, and down-regulation of let-7b in TAMs leads to changes in expression profiles of inflammatory cytokines, such as IL-12, IL-23, IL-10 and TNF-α. As a result, TAMs treated with let-7b inhibitors reduce angiogenesis and prostate carcinoma (PCa) cell mobility. Let-7b may play a vital role in regulating macrophage polarization, thus modulating the prognosis of prostate cancer.


Results
Generation and characterization of PCa-conditioned TAMs. Macrophages are highly plastic cells that respond to a variety of environmental cues by changing their phenotype and function 18 . Proinflammatory M1 macrophages promote T helper (Th) 1 responses and show tumoricidal activity. M2 macrophages display regulatory functions in tissue repair and remodelling and promote Th2 immune responses 19 . TAMs, key orchestrators in the tumor microenvironment, resemble M2-polarized macrophages. Circulating monocytes, which can pass through the vascular endothelium to mature into macrophages in the peripheral tissues, are activated in various ways by endogenous and exogenous factors 20 . To investigate if exposure to PCa tumor microenvironment can affect monocyte differentiation, we incubated human blood monocytes (isolated from healthy male donors) with culture medium (CM) collected from PCa cells for 7 days. Afterwards, we detected the phenotype of the macrophages. First, we analyzed expression of CD163, a marker for M2 in TAMs. TAMs expressed almost the same level of CD163 (78.3%) as M2 (79.9%) (Fig. 1A). Next, we measured the expression of IL-10, IL-12 and IL-1β which have been used for phenotyping macrophages. Our data show that TAMs displayed IL-10 high , IL-12 low , and IL-1β low phenotype (Fig. 1B). These data indicate that PCa-conditioned prostatic TAMs possess M2-like phenotype.

PCa-conditioned TAMs enhance tumorigenesis of PC-3 cells and promote angiogenesis of endothelial cells. In solid tumors, TAMs correlate with high vessel density and tumor progression.
Accumulated evidence demonstrates that TAMs play a critical role in the regulation of epithelial-mesenchymal transition in cancer. To validate the role of PCa-conditioned TAMs, we studied their effect on PC-3 cell proliferation, migration and invasion. The results show that PCa-conditioned TAMs significantly enhance PC-3 cell proliferation ( Fig. 2A), migration (Fig. 2B) and invasion (Fig. 2C). We also determined their ability to promote angiogenesis. As shown in Fig. 2D, prostatic TAMs were pro-angiogenesis, a powerful provocation compared to other macrophage subtypes, although they showed characteristics of M2-like phenotype.
Up-regulation of miRNA let-7b in PCa-conditioned prostatic TAMs. To investigate the mechanism by which TAMs could affect cytokine expression, we first detected the expression of let-7b. We found that prostatic TAMs significantly increased expression levels of let-7b compared with other macrophages (Fig. 3A). Next, we transfected TAMs with either let-7b inhibitors or negative control of inhibitors. We observed that let-7b was significantly down-regulated by as much as 37.12% with let-7b inhibitors but not with negative control of inhibitors (Fig. 3B), suggesting upregulated let-7b in PCa-conditioned TAMs can be efficiently suppressed by let-7b inhibitors.
Let-7b regulates expression of inflammatory cytokines in PCa-conditioned TAMs. TAMs, acting as tumorigenesis regulators, work partially through secretion of pro-inflammatory cytokines (such as TNF-α and IL-12) or anti-inflammatory cytokines (such as IL-10) 21 . To further investigate the role of let-7b in TAMs, we analyzed expression of inflammatory cytokines including IL-12, IL-23, IL-10 and TNF-α . Our results reveal that, in the presence of let-7 inhibitors, IL-12 and IL-23 were significantly up-regulated whereas TNF-α was down-regulated in PCa-conditioned TAMs. Interestingly, IL-10, a cytokine related to M2, was also significantly increased by let-7b inhibitors (Fig. 4). These data indicate that let-7b modulates the expression of IL-12, IL-23, IL-10 and TNF-α in PCa-conditioned TAMs.

Let-7b promotes mobility of PC-3 cells. Cell migration is essential for diffusion of cancer cells during
PCa progression 22 . We had observed that let-7b influences expression of inflammatory cytokines in TAMs. To clarify if introduction of let-7b inhibitors into TAMs could impair PCa migration, we incubated human PCa cells with CM from TAMs that had been transfected with let-7b inhibitors or negative control. In a cell migration assay, Scientific RepoRts | 6:25602 | DOI: 10.1038/srep25602 we observed that let-7b inhibitors significantly suppressed the capacity of PCa-conditioned TAMs to promote PC-3 migration as compared with negative control or TAMs without transfection (Fig. 5). These results suggest that let-7b expression in TAMs plays a critical role in promoting PCa migration.
Let-7b enhances pro-angiogenesis of PCa-conditioned TAMs. An important step during neo-angiogenesis is the formation and merging of tubes, produced by endothelial cells, to form a complex network of vessels and capillaries 23 . To determine the effect of let-7b on pro-angiogenesis of PCa-conditioned TAMs, we used human umbilical vein endothelial cells (HUVEC) as they have been reported to drive de novo angiogenesis in tube-like structure formation 24 . We treated HUVEC with CM from PCa-conditioned TAMs transfected with let-7b inhibitors, then assessed their ability to induce capillary morphogenesis. As shown in Fig. 6, let-7b inhibitors significantly suppressed tube-like structure formation, suggesting let-7b has a key role in driving vascularization of PCa.

Figure 2. TAMs promote proliferation, mobility and invasiveness of PC-3 cells and promote angiogenesis.
(a) Proliferation of PC-3 cells. PC-3 cells were exposed to CM from M0, M1, M2 and TAMs or 1640 medium as control for 72 h. Viability of PC-3 cells was measured by MTT assay. (b) Mobility of PC-3 cells. A line of PC-3 cells was scraped away in each well using a pipette tip after 6 h of serum starvation. Subsequently, cells were treated with CM from M0, M1, M2 and TAMs for 24 h. Migrated cells were observed from three randomly chosen fields (original magnification, 100× ) and the number of migrated cells was quantified by manual counting. (c) Invasiveness of PC-3 cells. PC-3 cells were loaded into the upper compartments and then placed into 24-well culture dishes containing different CM from M0, M1, M2 and TAMs, or RPMI 1640 medium as control. After 24 h of incubation at 37 °C, cells that migrated to the bottom of the membrane were stained with hematoxylin-eosin and counted using an inverted microscope (original magnification, 100× ). (d) Tube-like structure formation in HUVEC. HUVEC were seeded to the matrigel-coated plates, followed by addition of CM from M0, M1, M2 or TAMs, respectively. The effects on the morphogenesis of endothelial cells were recorded after 5 h with an inverted microscope equipped with CCD optics and a digital analysis system. Results were quantified by measuring the joint or vessel numbers in the field (original magnification, 100× ). *P < 0.05; **P < 0.01; ***P < 0.001. Scientific RepoRts | 6:25602 | DOI: 10.1038/srep25602 Discussion Let-7 family members, specifically let-7b, have been implicated as tumor suppressors in several types of human tumor cells including prostate carcinoma 25 . However, little has been reported on the role of let-7b in prostatic TAMs. In this study, we demonstrate the importance of let-7b, confirming that its decreased expression inhibits the pro-angiogenic effect of TAMs and their capacity to enhance PC-3 cell motility. The expression level of let-7b significantly and positively correlates to the level of TNF-α , while let-7 negatively regulates the expression of IL-10, IL-12 and IL-23 in TAMs.
miRNAs are universal regulators of differentiation, activation and polarization of macrophages. A number of studies have implicated different miRNAs in human monocytes/macrophages in response to inflammatory stimuli 26,27 . Our former studies had demonstrated that the level of let-7a/b/c/e was upregulated in TAMs as comparing with other macrophages, and among let-7 miRNAs, let-7b showed the most obvious difference (unpublished data). However, the role of let-7b in regulating macrophage polarization has been largely undefined. We found that let-7b is expressed in prostatic TAMs at the highest level, comparable to M0, M1 and M2 macrophages. Our data show that let-7b is involved in macrophage polarization and affects function of TAMs. Down-regulation of let-7b in TAMs significantly suppresses PCa migration and the function of pro-angiogenesis. One plausible explanation is that let-7b regulates a variety of inflammatory cytokines, leading to the change in biological properties of TAMs. Known relevant targets of let-7b are molecules involving cell cycle control with respect to differentiation and tumorigenesis like Estrogen Receptor-α 36, HMGA1, EZH2 and Cdc34 16,[28][29][30] . Recent studies found that let-7 regulates C/EBP-δ , an important transcriptional factor that has been shown to be required for a sustained TLR4 signals which induced NF-κ B and AP-1 activation 31,32 . Targeting of the SOCS4 3' untranslated region by let-7b resulted in translational repression and inhibition of SOCS4 promoted phosphorylation of STAT3 and STAT6 33 . These signal molecules regulate the secretion of a large number of cytokines such as IL-6 and IL-12. Experimental data indicate that the let-7 family members suppress several important immune-related genes including IL-6, IL-13 and IL-10 [34][35][36] . In support of this theory, our data show that transfection with let-7 inhibitors can change pro-and anti-inflammatory cytokine profiles.
All solid tumors require the induction of new blood vessels to grow, and angiogenesis is associated with tumor growth and metastasis. Microvessel density in the area of the most intense neovascularization in invasive, early-stage breast carcinoma is an independent and highly significant prognostic indicator for overall and relapse-free survival in patients 37 . At least two general categories are recognized: (i) angiogenic activity arises from the tumor cell itself by releasing angiogenic molecules such as basic fibroblast growth factor; (ii) angiogenic activity arises from host cells recruited by the tumor (e.g. macrophages) 23 . Cell motility is a fundamental component of many physiological and pathological processes and drives disease progression in cancer 22 . It is a critical step in the progression of PCa to the metastatic state, the lethal form of the disease.
TAMs have been associated with enhanced tumor progression, including cancer cell growth and spread, angiogenesis and immune suppression. In this study, human monocytes became prostatic TAMs after incubation with CM collected from PC-3 cell culture. The resultant TAMs displayed characteristics of M2-like macrophages, such as CD163 high , IL-10 high and IL-12 low . We found that TAMs significantly enhance PC-3 cell proliferation when compared to control, M0, and M1. Moreover, we found that transfection of TAMs with let-7 inhibitors decreases the level of let-7b and reverses the effect of TAMs on PCa migration and angiogenesis.
Cytokines in the tumor microenvironment have the capacity to pilot recruitment, maturation and differentiation of infiltrating leukocytes, playing a vital role in the growth and metastasis of tumor cells. Our data demonstrate that IL-10, IL-12 and IL-23 are significantly up-regulated in TAMs treated with let-7b inhibitors, whereas TNF-α shows significant down-regulation. IL-12 and IL-23 are predominantly pro-inflammatory/pro-stimulatory cytokines that have key roles in the development of Th1 and Th17 cells, respectively 38 . Considering the M1 phenotype produces IL-12 and IL-23, we hypothesized that TAMs treated with let-7b inhibitors would display partial characteristics of M1 with IL-12 high and IL-23 high phenotype. Accumulated evidence indicates that IL-12 is a cytokine with both immunostimulatory and antiangiogenic effects [39][40][41][42] . The mechanism of IL-12's antitumor action may depend, not only on the immunostimulatory activity of this cytokine, but also on its effect on tumor cell angiogenesis 43 . IL-10 was also significantly up-regulated in TAMs treated with let-7b inhibitors. One explanation is that IL-10 is a target of let-7b, and its expression is negatively mediated by let-7b 35 . With decreased expression of let-7b, mRNA stability of IL-10 increases in TAMs treated by an inhibitor. Although IL-10 has been reported as a cytokine related to M2, IL-10 is drawing attention as an inhibitor of tumor angiogenesis 44 . Kohno and colleagues have reported anti-angiogenic and tumor suppressive effects of IL-10 in ovarian cancer cells 45 . In addition, IL-10 also inhibits cell mobility 46,47 . In our data, down-regulation of let-7b leads to significant up-regulation of IL-10, IL-12 and IL-23 in TAMs. We speculate that these cytokines synergize to suppress angiogenesis. We also found that TNF-α is significantly down-regulated in TAMs treated with let-7b inhibitors. TNF superfamily cytokines are increasingly recognized as key modulators of angiogenesis. TNF-α is not only involved with Figure 6. Down-regulation of let-7b suppresses pro-angiogenic effect of TAMs. HUVEC were plated in a 96-well plate pre-coated with matrigel, followed by the addition of CM from TAMs treated with let-7b inhibitors, negative control (NC), or RPMI 1640 medium (control) for 5 h. The effects on the morphogenesis of endothelial cells were imaged with an inverted microscope equipped with CCD optics and a digital analysis system. Results were quantified by measuring the joint or vessel numbers in the field (original magnification, 100× ). *P < 0.05; **P < 0.01.
In summary, our results indicate that the tumor-promoting role of prostatic TAMs may be partially ascribed to up-regulation of let-7b, which regulates expression of IL-12, IL-23, IL-10 and TNF-α . The expression profiles of these inflammatory cytokines partially affect TAM function. TAMs that were treated with let-7b inhibitors had reduced angiogenesis and PCa cell mobility. It should be noted that let-7b may possess various functions owing to its pleiotropic regulation of genes. It is our expectation that additional let-7b target genes will be identified in the near future. Our findings suggest that let-7b is a promising modulator for macrophage polarization. Further studies are required on the origin of let-7b up-regulation and its signal pathway to determine if let-7b can be used to mediate macrophage polarization.

Materials and Methods
Isolation and culture of human peripheral blood macrophages. Blood monocytes were isolated from healthy donor buffy coats. Peripheral blood mononuclear cells (PBMCs) were isolated using a Ficoll (Solarbio Life Sciences, Beijing, China) density gradient, and subsequently monocytes were isolated from PBMCs using anti-CD14 magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocol. Non-adherent cells were removed, and purified monocytes were incubated for 7 days in RPMI 1640 (Life Technologies Corporation, Grand Island, NY, USA), supplemented with 10% FBS (Life Technologies, Burlington, ON, Canada) and 50 ng/ml M-CSF (Peprotech, Rocky Hill, NJ, USA) to obtain macrophages. M0 cells were obtained by treating with serum-free medium for 48 h. M1 macrophages were polarized by stimulating overnight with 100 ng/ml lipopolysaccharides (Peprotech) and 100 ng/ml IFN-γ (Peprotech). M2 macrophages were polarized by stimulating overnight with 20 ng/ml IL-4 (Peprotech). Prostatic TAMs were obtained by culturing monocytes for 7 days in RPMI 1640 10% FBS with 50% of conditioned medium (CM) from PC-3 cells. Before CM was obtained from PC-3 cells, M0, M1, M2 and TAMs, these cells were incubated for 48 h in serum-starved condition. Then CM was harvested, clarified by centrifugation and used freshly. The study was approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University, and written informed consent was obtained from all donors. All experimental protocols were in accordance with the approved guidelines for safety requirements of Jiangxi Academy of Medical Sciences, Nanchang University. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. PC-3 cell viability was measured by MTT assay. Briefly, PC-3 cells were seeded at 5 × 10 3 cells/well in 96-well plates, and allowed to adhere to obtain 80% confluent monolayer. The medium was replaced with CM from different cells. After 72 h incubation, cell growth was measured at 490 nm using SpectraMax M4 Multimode Microplate reader (Molecular Devices). The number of viable cells was presented relative to control group.

RNA extraction and real-time reverse transcription PCR. Total RNA was extracted using Invitrogen
Trizol Reagent (Life Technologies Corporation). For miRNA quantification, 100 ng total RNA was reverse transcribed directly using stem-loop primers 53 . For mRNA analyses, cDNAs were synthesized from 2 μ g total RNA, using oligo (dT) 15 primers and Moloney Murine Leukemia Virus Reverse Transcriptase (Life Technologies Corporation). Quantitative real-time PCR was performed using the SYBR Green PCR Master Mix (Tokara, Kyoto, Japan) in a final volume of 20 μ L on Bio-RAD CFX96 TM Real-Time System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The expression of miRNA and mRNAs was normalized to U6 and GAPDH, respectively. Data are presented as relative quantification based on the calculation of 2 −ΔΔCt . All primers used in this study are shown in change to Supplementary Dataset. Matrigel angiogenesis assay. 50 μ l of matrigel was added to each well of a 96-well plate, then placed in a incubator at 37 °C for 30 min. HUVEC (ATCC) (2 × 10 4 cells/well) were added to the matrigel-coated plates in a final volume of 100 μ l. The effects on morphogenesis of endothelial cells were recorded with an inverted microscope equipped with CCD optics and a digital analysis system (Olympus, Tokyo, Japan) 5 h later. Results were quantified by measuring the joint or vessel numbers in the field.