Background
Hepatocellular carcinoma (HCC) is the sixth most common cancer and third leading cause of cancer-related death worldwide [
1]. Treatment of HCC has changed greatly in recent years, but hepatic resection and transplantation are still considered the main curative therapy, and a high postoperative recurrence rate is a major impediment to prolonging patient survival [
2].
Crosstalk between tumor cells and their microenvironment underlies the pathogenesis of HCC [
3]. Transplantation of normal hepatocytes into a neoplastic-prone liver microenvironment delays the growth of hepatic nodules and the emergence of HCC [
4]. A unique immune/inflammation response signature is associated with HCC intrahepatic metastasis, and inflammatory cytokines can predict poor survival of patients with HCC [
5,
6]. These findings suggest that the immune/inflammation microenvironment may foster the development of HCC. Tumor-associated macrophages (TAMs) are key components of the cancer microenvironment [
7,
8]. TAMs originate from circulating monocyte precursors that are recruited to the tumor by tumor-derived signals, including chemokine (C-C motif) ligand 2 (CCL2) and macrophage colony-stimulating factor (M-CSF). In response to distinct microenvironment signals, macrophages can exert either anti- or pro-tumor activities [
9], classified as M1 (or classical) or M2 (or alternatively activated), respectively [
10,
11]. In HCC, most macrophages in the peritumor region exhibit an M2 phenotype, which is probably determined by the tumor cell [
12,
13]. These macrophages facilitate tumor growth, metastasis, and angiogenesis and are associated with poor patient survival [
13‐
16]. In many types of tumors, M-CSF is an essential regulator or recruiter of macrophages and is mainly produced by tumor cells [
17,
18]. After binding to colony-stimulating factor 1 receptor (CSF-1R) on macrophages, M-CSF can activate macrophages, promoting secretion of growth factors that are essential for the pre-metastatic niche and tumor growth or metastasis [
19‐
21]. Some studies have also found high expression of M-CSF and its receptor in peritumoral liver tissue, which is associated with poor survival of patients with HCC after curative resection [
6,
22].
microRNAs (miRNAs) are a class of 22-nucleotide noncoding RNAs. The aberrant expression of specific miRNAs is directly involved in tumorigenesis, including growth, apoptosis, angiogenesis, and metastasis [
23‐
27] and affects the biology of cellular components belonging to the tumor microenvironment, including endothelial cells, pericytes, fibroblasts, and immune cells [
28‐
31]. In HCC, miRNAs have been reported to regulate the biology of endothelial cells and Treg cells [
32,
33]. It has also been reported that miR-214 modulates macrophage polarization in HCC [
34]. However, no study has addressed whether miRNAs regulate M-CSF expression in tumor cells and the recruitment of macrophages in HCC.
Deregulation of miR-26a may function as either a tumor suppressor or promoter. miR-26a is down-regulated in breast cancer, anaplastic carcinomas, and oral squamous cell carcinoma [
35‐
38] but up-regulated in cholangiocarcinoma and glioma [
39‐
41]. In HCC, a reduced level of miR-26a is associated with poor overall survival of patients with HCC [
42]. Patients with hepatitis B virus-related HCC had a lower level of miR-26a in blood compared with patients with chronic hepatitis B [
43]. In addition, miR-26a can inhibit cancer cell proliferation and protect against disease progression without toxicity in an HCC mouse model [
44‐
46]. The underlying mechanisms might be that miR-26a can inhibit tumor growth, metastasis, and angiogenesis in HCC [
27,
44,
47,
48]. However, no study has addressed the role of miR-26a in TAMs.
In the present study, we investigated whether miR-26a regulates recruitment and function of macrophages in HCC and the underlying mechanisms of miR-26a.
Discussion
miR-26a has been reported to be able to reduce proliferation, metastasis, and angiogenesis in HCC [
27,
44,
47,
48]. We further demonstrated that miR-26a can regulate the recruitment of macrophages. Some studies have reported miRNAs associated with macrophage infiltration, apoptosis, and phenotype. For example, let-7d suppresses macrophage infiltration by targeting COL3A1 and CCL7 [
52], miR-142-5p can regulate apoptosis of macrophages by targeting TGF-β2 [
53], and miR-155 can modulate macrophage polarization by targeting C/EBP-β [
54]. Here, we first demonstrated that miR-26a can suppress macrophage recruitment by regulating M-CSF expression in HCC.
In our previous study, we found that miR-26a expression was lower in HCC tumor samples than in paired noncancerous liver tissue, and patients with lower miR-26a expression had shorter overall survival compared with patients with higher miR-26a expression [
42]. The underlying mechanism related to miR-26a may be suppression of HCC cell proliferation and metastasis by regulation of the IL-6-stat3 signaling pathway and inhibition of angiogenesis in HCC by down-regulating Akt/VEGF signaling [
27,
47,
48]. In addition, Kota et al. [
44] reported that miR-26a can inhibit cancer cell proliferation in an HCC mouse model by targeting cyclin D2 and E2. However, the underlying mechanisms for how miR-26a affects the survival of patients with HCC are still not fully understood. The present study was designed to show a new role of miR-26a in the recruitment of macrophages in HCC.
M-CSF is one of the major cytokines controlling the proliferation, differentiation, and functional regulation of macrophage [
55]. The expression level of M-CSF has been found to be associated with higher histological tumor grading, more frequent metastases, and poor prognosis in many cancer types, including papillary renal cell carcinoma, serous and mucinous ovarian epithelial tumors, endometrioid carcinomas, breast cancer, and especially HCC [
51,
56‐
59]. Our previous study showed that high expression of M-CSF in peritumoral liver tissue is associated with poor survival after curative resection of HCC [
6,
22]. Various miRNAs have been reported to be involved in the regulation of M-CSF expression. Mandal et al. [
49] found miR-21a can induce M-CSF expression by regulating the PI3K/Akt signal pathway. Cimino et al. [
60] reported M-CSF is a direct target of miR-148b. Wang et al. [
61] identified M-CSF as a target gene of miR-214, and Zhang et al. [
62] reported M-CSF as a target of miR-128. The present study found miR-26a regulates M-CSF expression and recruitment of macrophages. In addition, M-CSF also plays an important role in the polarization of macrophages. It can stimulate macrophages and induce them to exhibit an anti-inflammatory M2-type of activation, which produces higher IL-12/23 but lower CCL17, CCL22, and IL-10 [
50,
51,
63,
64]. The present study also found miR-26a can induce a pro-inflammatory M1-type of activation for macrophages and may affect the overall survival of patients with HCC partly through reducing macrophage recruitment and down-regulating M-CSF expression.
This study has some potential limitations. We found miR-26a can inhibit macrophage recruitment by down-regulating M-CSF expression, but recruitment might be influenced by many factors. Our preliminary study showed miR-26a expression did not affect expression of CCL2 and GM-CSF (data not shown), which are two major cytokines regulating recruitment of macrophages [
51,
65].
M-CSF inhibitor has been reported to impede macrophage recruitment in malignancy [
66]. For HCC, zoledronic acid, a bisphosphonate with a macrophage-modulating effect, can inhibit growth of HCC cells and delay disease progression of bone metastases [
67,
68]. Our previous study reported that depletion of macrophages using zoledronic acid can enhance the effect of sorafenib in HCC as well [
69]. With all data considered together, M-CSF and macrophage might be potential therapeutic targets for HCC. Our data provide evidence that miR-26a may suppress the recruitment of macrophages by down-regulating M-CSF expression. The data also suggest that miR-26a may be a marker for the grade of malignancy in HCC as well as a potential therapeutic target in patients with HCC.
Methods
Cell lines
The HCC cells which were altered expression levels of miR-26a by using recombinant lentivirus vector (Genechem, Shanghai, China) were established in our previous study. Human monocyte cell line THP-1 were obtained from Shanghai Institute of Cell Biology (Shanghai, China). The HCC cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA) containing 10 % fetal bovine serum (FBS), and THP-1 cells were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10 % FBS. All cells were maintained in a humidified incubator at 37 °C with an atmosphere of 5 % CO2.
Collection of the CM
HCC cells were treated with LY294002 (20 μM, Beyotime, Jiangsu, China) or vehicle for 12 h, then incubated in DMEM with 0.1 % BSA for 24 h. The CM was centrifuged for 20 min at 3000 rpm, and the resultant pellet was stored at −80 °C. In the M-CSF blocking assay experiments, neutralizing antibody against M-CSF (R&D Systems, Minneapolis, MN) and control IgG were added to CM 30 min before further experiments.
Quantification of M-CSF in the CM
ELISA (R&D Systems) was used to measure M-CSF concentrations in the CM. The total protein concentration was measured by bicinchoninic acid (BCA) assay, and the M-CSF concentration was normalized according to the total cellular protein.
Cell migration assay
Chambers (Corning, Tewksbury, MA) with 8.0-μm polycarbonate filter inserted in 24-well plates were used in the quantitative cell migration assays as described before [
27]. THP-1 cells stimulated by PMA (1 × 10
5 cells/well) were added in the upper chamber, and the lower chamber was filled with the previously collected CM. The 24-well plates filled with cells were placed in a thermostatic incubator at 37 °C for 16 h. Thereafter, the migrated cells were fixed with methanol, stained with crystal violet, and photographed under an inverted microscope. The areas of stained cells from three random fields at × 200 magnification were assessed by using Image-Pro Plus software (Media Cybernetics Inc, Bethesda, MD).
Western blot assay
As described in our previous study [
27], cells were lysed in buffer (150 mM NaCl, 50-mM Tris-HCl, pH 8.0, 0.1 % SDS, 1 % Triton X-100) containing protease and phosphatase inhibitors. Fifty micrograms of whole cell extracts were subjected to SDS-PAGE gel and transferred to nitrocellulose membranes. The membranes were blocked with 5 % nonfat milk for 2 h and then incubated with respective primary antibody overnight at 4 °C, followed by incubation with the appropriate HRP-conjugated secondary antibody for 2 h at room temperature. Blots were visualized with an ECL detection kit (Pierce, IL) and analyzed using Quantity One 1-D Analysis Software (Bio-Rad, San Francisco, CA).
Real-time PCR assay
Equal amounts of THP-1 cells stimulated by PMA were treated with the previously collected CM. After 48 h, total RNA was extracted using TRIzol Reagent (Sigma, St. Louis, MO). As described before [
69], reverse transcription reactions and quantitative real-time PCR were performed using RT-PCR kit (TaKaRa, Shiga, Japan). The primers used for the amplification of human genes are listed as follows: IL-12 (5′-TGCCCATTGAGGTCATGGTG-3′ [forward]; 5′-CTTGGGTGGGTCAGGTTTGA-3′ [reverse]), IL-23 (5′-CAGAGAGAATCAGGCTCAAAGC-3′ [forward]; 5′-AGCAACAGCAGCATTACAGC-3′ [reverse]), CCL17 (5′-CTGGGACCTCCACCGTT-3′ [forward]; 5′-CTCACTGTGGCTCTTCTTCGT-3′ [reverse]), CCL22 (5′-AAACTAATGTCCCTCCCCTCTC-3′ [forward]; 5′-TTTGGGGCTTCACATTGACC-3′ [reverse]), IL-10 (5′-TGCCTAACATGCTTCGAGATC-3′ [forward]; 5′-CCAGGTAACCCTTAAAGTCCTC-3′ [reverse]), and ACTB (5′-ACTGGGACGACATGGAGAAAATC-3′ [forward]; 5′-CTCGCGGTTGGCCTTGG-3′ [reverse]).
Xenograft model of human HCC in nude mice
As described in our previous study [
27], male BALB/c nude mice (5 weeks old) were purchased from the Shanghai Institute of Materia Medica, Chinese Academy of Science, and housed under specific pathogen-free conditions. The experimental protocol was approved by the Shanghai Medical Experimental Animal Care Commission. Twenty mice were randomized into four groups, and various cancer cells (6 × 10
6 cells) in 200 μl of normal saline were implanted by subcutaneous injection to obtain subcutaneous tumors. Tumor dimensions were measured by vernier caliper every 4 days, and the mice were killed after 4 weeks. After final measurement, the tumors were placed in 4 % paraformaldehyde solution. The tumor volume was calculated according to the formula: tumor volume = (largest diameter × perpendicular height
2)/2.
Immunohistochemical assay
Paraffin-embedded tumor tissues from animal Immunohistochemistry was performed on the sections or tissue arrays containing tumor tissues from patients with HCC who had antibodies against M-CSF (1:100; Abcam, Cambridge, MA) or CD68 (1:100; Abcam, Cambridge, MA). The integrated optical density (for M-CSF) or area (for CD68) of positive staining/total area was quantified by Image-Pro Plus software [
27]. For the reading of each antibody staining, a uniform setting for all the slides was applied.
Patients and follow-up
HCC specimens used in the immunohistochemical assay were obtained from patients who received radical resection between 1999 and 2003 at the Liver Cancer Institute and Zhongshan Hospital (Fudan University, Shanghai, China); these specimens have been described previously [
27,
42]. None of the patients received any preoperative anticancer treatment. The research was approved by the research ethics committee of Zhongshan Hospital. A total of 80 cases were used to examine CD68 expression, and 52 cases were used to examine CD68 expression. The miR-26a expression data were collected in our previous study. All the patients were followed up until 2011 with a median observation time of 60.8 months. This study was approved by the Zhongshan Hospital Research Ethics Committee. All patients provided their written informed consent to participate in this study.
Statistical analysis
Data were analyzed using SPSS 18.0 (SPSS, Inc.). Quantitative variables were analyzed using the unpaired two-tailed Student’s t test and Spearman correlation test. P < 0.05 was considered statistically significant.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
ZTC, XDZ, and JYA performed the experimental work. WQW, DMG, JK, NZ, YYZ, BGY, and DNM participated in the experiments. XDZ and HCS conceived the study and participated in its design and coordination. The manuscript was written by ZTC, XDZ, and JYA. All authors read and approved the final manuscript.