Long non-coding RNAs (lncRNAs) are RNAs that are longer than 200 nucleotides and do not code for proteins [1
]. Thousands of lncRNAs have been discovered to date. By binding to DNA, mRNAs, miRNAs or target proteins, lncRNAs can regulate the transcription, processing, translation, turnover, and protein activity of other protein coding genes [2
]. LncRNAs are found to play important roles in regulating genomic imprinting, cell cycle, cell differentiation, tumorgenesis, metastasis and a variety of biological processes [3
]. Accumulating evidence also emerge to suggest lncRNA function in both innate and adaptive immunity including their regulation of the development, activation, and homeostasis of the immune system [6
Exosomes are small extracellular vesicles and are composed of a lipid bilayer ~50 to 100 nm in diameter. They are secreted from multivesicular endosomes by many different cell types and exist in almost all biological fluids as well as in culture medium of most cell types [7
]. Exosomes contain a large variety of biological components including proteins, mRNAs, miRNAs [8
], and lncRNAs [9
] and hold great promise as novel biomarkers for clinical diagnosis. Both in vivo and in vitro studies have demonstrated that exosomes can be taken up by target cells and play important roles in cell-cell communication by transferring the molecular constituents to target cells [10
]. A substantial body of literature has implicated exosomes–mediated transfer of lncRNAs in regulating immune response, cellular stress responses, chemosensitivity, tumor cell growth, and adhesion [11
Macrophages are critical players in the innate immune defense and are increasingly appreciated as participants in the tumor microenvironment. Macrophages are a functionally heterogeneous cell population that hold a high degree of plasticity. In response to stimulation in the microenvironment, macrophage can be phenotypically polarized to two main groups: classical (or M1) and alternative (or M2) macrophages. M1 macrophages, which can be induced in vitro by lipopolysaccharide (LPS) and interferon γ (IFN-γ) elicit rapid proinflammatory responses, pathogen clearance, and display anti-tumor activity [15
]. M2 macrophages, which can be induced in vitro by interleukin 4 (IL-4) and IL-13 show anti-inflammatory and tissue reparative activities, can promote tumorgenesis and tumor progression [16
]. The switch from a proinflammatory (M1) to an anti-inflammatory phenotype (M2) is usually observed in tumor-associated macrophages during tumor progression from the early stage to a more advanced stage [17
Previously, lncRNA TUC339 was identified and found to be enriched in HCC-derived exosomes [18
]. Exosomal transfer of TUC339 to neighbor tumor cells promotes HCC cell proliferation and reduces cell adhesion to extracellular matrix. In this case, we ask whether HCC-derived exosomal lncRNAs can be transferred to neighbor macrophages and how this intercellular communication would influence macrophage function. We show that HCC-derived exosomes can indeed target neighbor macrophages and subsequently transfer TUC339 and other lncRNAs to macrophages. Gain and loss-of-function studies suggest TUC339 modulates macrophage cytokine production, phagocytosis, and M1/M2 polarization. These findings provide new mechanistic insights into exosomes as mediators in the tumor microenvironment and lncRNAs act as modulators of macrophage polarization, which advances our knowledge towards a tumor-immune interaction.
In this study, we identified lncRNA TUC339 as a novel signaling mediator that is enriched in HCC-derived exosomes and plays important roles in macrophage activation and M1/M2 polarization regulation. We showed that over-expression of TUC339 in THP-1 cells resulted in reduced pro-inflammatory cytokine production, decreased co-stimulatory molecule expression, and compromised phagocytosis and vice versa. Moreover, TUC339 expression was positively associated with M(IL-4) macrophages polarization. Blockade of TUC339 expression in macrophages inhibited M(IL-4) polarization while excess TUC339 expression in macrophages promoted M(IL-4) polarization. Our results unveil a novel function of HCC-derived exosomal lncRNA in the tumor microenvironment and a novel mechanism of tumor-immune cells interaction.
Previously, lncRNA TUC339 was identified as a lncRNA that is enriched in HCC-derived exosomes and transfers between HCC cells to promote tumor growth and spread [18
]. In this scenario, we provide evidence to suggest TUC339 as a novel signaling mediator, which can be transferred from HCC tumor cells to environmental macrophages and subsequently dampen the immune response to tumor cells. As the tumor progresses, tumor cells develop a variety of strategies to evade immune surveillance and interrupt the immune attack [21
]. For example, tumor-derived exosomes are enriched for FasL and TRAIL, which can promote T-cell apoptosis [22
]. Along with these findings, our result provides important new insights into tumor-immune interaction and uncovers potential therapeutic targets for treatment of HCC.
Phagocytosis is a major feature of macrophages that is meant to engulf and clear external microbes, dead or damaged cells to provide an effective immune defense, and maintain homeostasis of the organism [24
]. Phagocytosis can be effectively triggered by a toll-like receptor (TLR), a Fcγ receptor (FcγR), and complement receptor (CR) mediated ligand binding. FcγR facilitates the phagocytosis of particles that are opsonized by antibodies. In our microarray study, we showed that the FcγR-mediated phagocytosis pathway was up-regulated upon knockdown of TUC339, which may partially explain the enhanced phagocytosis observed in TUC339 knockdown. As known, the actin cytoskeleton, which maintains cellular morphology, is the primary force driving phagocytosis [25
]. We observed a down-regulation of the actin cytoskeleton pathway upon the over-expression of TUC339, which is in line with down-regulated phagocytosis under that condition. Moreover, GO analysis of the microarray data indicates the cell motility and cell migration of THP-1 cells, which were suppressed upon over-expression of TUC339. Further exploration on direct downstream targets of TUC339 and more detailed mechanistic analyses on TUC339 effect on phagocytosis are required in future studies.
Inflammation is a defensive response of our immune system against microbial invasion or tissue damage [26
]. LPS-induced inflammation promotes the classical activation pathway of macrophages toward M1 phenotype (pro-inflammatory) through TLR (toll-like receptor) stimulation, which results in pro-inflammatory cytokines production such as TNF-α, IL-1β, IL-6, IL-12, and IL-23. In this case, upon TUC339 knockdown, we observed increased pro-inflammatory cytokine production, enhanced phagocytosis, and elevated co-stimulatory molecule expression by THP-1 cells, which suggests classical activation of macrophage. Over-expression of TUC339, in contrast, led to reduced pro-inflammatory cytokine production, compromised phagocytosis, and decreased co-stimulatory molecule expression by THP-1 cells. These observations are consistent with each other and lead us to examine the macrophage polarization status upon alteration of TUC339 expression.
Macrophages can be polarized to pro-inflammatory M(IFN-γ + LPS) or anti-inflammatory M(IL-4) status. These two distinct subgroups can be dynamically converted into each other under a specific microenvironment [27
]. Previous studies revealed a significantly altered lncRNA and mRNA expression profile in macrophages exposing to different activating conditions, which indicated that lncRNAs may play important roles in regulating macrophage polarization. LncRNA TCONS_00019715 was found to play an important role in promoting macrophage polarization to the M(IFN-γ + LPS) phenotype [28
]. LncRNA cox-2 can inhibit tumor growth by inhibiting the polarization of M2 macrophages [29
]. Exosomes have also emerged as important signaling mediators of macrophage polarization. LPS-preconditioned mesenchymal stromal cells-derived exosomes induced up-regulation of anti-inflammatory cytokines and M2 macrophage activation [30
]. Tumor-derived exosomal miRNA can be transferred to tumor-associated macrophages and stimulate polarization towards the M2 phenotype to promote tumor progress [31
]. In this study, to examine whether TUC339 contributes to the plasticity of macrophage polarization, we measured the expression of TUC339 in M(IFN-γ + LPS) and M(IL-4)-polarized activation of macrophages and found that M(IL-4) macrophages exhibited a considerably higher level of TUC339 than control and M(IFN-γ + LPS) macrophages. Next, we examined differential expression of TUC339 during macrophage polarization by incubating M(IFN-γ + LPS) macrophages with IL-4 and M(IL-4) macrophages with IFN-γ + LPS. Our experiments demonstrated that M(IFN-γ + LPS)-to-M(IL-4) conversion resulted in increased TUC339 expression while M(IL-4)-to-M(IFN-γ + LPS) led to decreased TUC339 expression. Lastly, it was very interesting to see that knockdown of TUC339 stimulated M(IL-4)-to-M(IFN-γ + LPS) transition (Figure 6
g–j) while over-expression of TUC339 stimulated M(IFN-γ + LPS)-to-M(IL-4) transition (Figure 7
g–l). Notably, both THP-1 and U937 human macrophage cell lines were tested and similar conclusions were drawn, thus arguing against the possibility that the regulatory effect of TUC339 was cell line-specific. Taken together, our data suggest that exosomal TUC339 plays an important role in promoting macrophage polarization to the M(IL-4) phenotype. From our microarray data, we found that TUC339 is involved in cytokine receptor signaling pathways and CXCR chemokine receptor binding pathways, which may provide some clues on the underlining mechanisms of this regulation even though the exact details are worth further investigation.
In summary, the data presented in this study demonstrate a novel role for HCC-derived exosomal lncRNA TUC339 in macrophage activation and M1/M2 polarization. Our study not only advanced our understanding of lncRNA on regulating macrophage function but also shed light on the complicated interactions between tumor cells and innate immunity.
4. Materials and Methods
4.1. Cell Culture
THP-1, U937, and HL-7702 cells (ZSGB-BIO, Beijing, China) were cultured in RPMI-1640 medium (HyClone Laboratories, Logan, UT, USA) containing 10% heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (HyClone Laboratories, Logan, UT, USA). Cells were cultured at 37 °C in a humidified incubator in which the concentration of CO2 was 5% and were used in the exponential growth phase.
4.2. Isolation of Exosomes Derived from PLC/PRF/5 Cells
PLC/PRF/5 cells (1 × 106/well) were plated in vesicle-depleted medium for 2 days prior to the collection of exosomes. The medium was first centrifuged at 300× g for 10 min and then at 2000× g for 20 min at 4 °C to remove cells and then at 10,000× g for 20 minutes at 4 °C. The supernatant was then centrifuged at 100,000× g for 70 min at 4 °C to pellet exosomes, which were then washed by resuspending in phosphate-buffered saline (PBS) and ultra-centrifuged at 100,000× g for 70 min 4 °C. The final pellet was re-suspended with 50 to 100 uL PBS and stored at 4 °C or −80 °C.
4.3. Transmission Electron Microscopy
Pellets of exosomes were added to carbon film copper network and adsorbed 5 min to dry up. Then copper network was stained with phosphotungstic acid for 3 min and blotted with paper until air dried. The images were obtained using FEI Technai G2F20S-Twin filed-emission transmission electron microscopy (FEI-TEM).
4.4. Nanoparticle Tracking Analysis
The size distribution analysis of exosomes was determined by the Shanghai XP Biomed Ltd. using an Electrophoresis & Brownian Motion Video Analysis Laser Scattering Microscopy (Zataview, Particle Metrix, Germany). The capture settings and analysis settings were performed manually, according to the manufacturer’s instructions.
4.5. Western Blot
Cells were lysed with RIPA buffer (Sigma, St. Louis, MO, USA). Isolated exosomes were lysed in 5× protein loading buffer (Beyotime, Shanghai, China). Equivalent amounts of cell lysate and exosomes were subjected to 10% SDS-polyacrylamide gel and transferred to PVDF membrane (Millipore, Burlington, MA, USA). After being blocked with 5% skim milk for 1 h at room temperature, the membrane was probed with the following primary antibodies: CD63 (Abcam, Cambridge, MA, USA, 1:2000), β-Actin (Abcam, 1:2500), and the HRP-conjugated secondary antibody against rabbit (Abcam, Cambridge, MA, USA, 1:5000). The blot was visualized by enhanced chemiluminescence using ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA, USA).
4.6. Cellular Internalization of PLC/PRF/5 Cells-Derived Exosomes
PLC/PRF/5 cells-derived exosomes were labeled with PKH67 Green Fluorescent Cell Linker Mini Kit (Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer’s protocol with minor modifications. Exosomes diluted in PBS were added to 100 μL Diluent C. In parallel, 0.5 μL PKH67 dye was added to 100 μL Diluent C and incubated with exosomes solution for 4 min. To bind excess dye, 200 μL 1% BSA/PBS was added. The labeled exosomes were filtered using 0.22 μm filter to remove excess dye and added onto THP-1 cells cultured in a Thermo Scientific eight-chamber slide. After incubation for 24 h, cells were stained with Hoechst 33258 (Sigma-Aldrich, St. Louis, MO, USA) and examined by confocal fluorescence microscopy.
4.7. Macrophage-Differentiation and Polarization Conditions
THP-1 or U937 cells were treated with 100 ng/mL phorbol-12-myristate-13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO, USA) overnight. To induce the polarization of macrophages, PMA-differentiated THP-1 cells were treated with 20 ng/mL of IFN-γ and 100 ng/mL of LPS (Sigma-Aldrich, St. Louis, MO, USA) to achieve M(IFN-γ + LPS) polarization or with 20 ng/mL of IL-4 (PeproTech, Rocky Hill, NJ, USA) to achieve M(IL-4) polarization. The non-polarizing THP-1 macrophages were cultured and left untreated. After 18 h of polarization, the adherent cells were harvested for further analysis.
4.8. Phagocytosis Assay
THP-1 cells (1 × 106/well) were plated in six-well culture plates and allowed to adhere overnight. The Latex beads-rabbit IgG-FITC complex (Cayman, Ann Arbor, MI, USA) was added directly to the culture medium at a 1:200 dilution and incubated at 37 °C for two hours. Cells were gently washed with assay buffer twice, which was followed by visualization.
4.9. CCK-8 Assay
For cell viability studies, THP-1 cells were seeded (1 × 104/well) in collagen-coated 96-well plates in the appropriate media and incubated for 24 h. Then medium was replaced with medium containing 100 ng/mL PMA and incubated for 24 h. After being transfected with siRNA or plasmid for 48 h, THP-1 cells were treated with 1 μg/mL LPS for 24 h. The viability was assessed using CCK-8 solution (Dojindo Molecular Technologies, Rockville, MD, USA) and a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).
4.10. Real-Time PCR
Total RNAs were extracted from cells using Trizol (Thermo Fisher Scientific, Waltham, MA, USA) and 1 μg of RNA was reverse-transcribed to cDNA using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA). The expression of genes was quantified using SYBR green reagents (Life Technologies, Carlsbad, CA, USA) on a CFX96 touch real time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA) using primers indicated in Supplementary Table S1
4.11. Transfection of siRNA
Three independent siRNAs against TUC339 and lincRNA-VLDLR along with unrelated control siRNA (si-ctrl) were purchased from Ribobio (Guangzhou, China). THP-1 cells were transfected with 50 nM siRNA to lncRNA or si-ctrl using INTERFERin (Polyplus-transfection SA, Illkirch-Graffenstaden, France) for 48 h before further experiments. si-TUC339-1 was used in functional studies.
The levels of TNF-α and IL-1β in culture supernatants were determined by using ELISA kits (Wuhan Boster Bio-Engineering, Wuhan, China), according to the manufacturer’s instructions. The levels of TNF-α and IL-1β were quantified using a standard curve established by a serial dilution of standard concentration.
4.13. Northern Blot
A specific 5′-biotinylated probe to detect TUC339 was designed and synthesized by Tsingke (Chengdu, China) with the sequence shown below: 5′-GGATCGGTGTGAAATAACGGGCCCATATAAATCCC-3′. Additionally, 10 μg total RNAs were separated on 6% Urea PAGE gel and were transferred to a nylon membrane. Northern blotting was performed with the Chemiluminescent Nucleic Acid Detection Module (Thermo scientific, Waltham, MA, USA). The probed membrane was washed twice with 2 × SSC (300 mM NaCl, 30 mM sodium citrate, pH 7.0) with 0.1% SDS for 10 min at room temperature and with 0.1 × SSC with 0.1% SDS for 5 min. HRP-conjugated anti-digoxigenin was used to detect the hybridized probe by chemiluminescent imaging with a lumino-image analyzer.
4.14. Gene Over-Expression
Full length of TUC339 was cloned into the pTracer-CMV2 vector (Jingmai BioTech, Chengdu, China). Differentiated THP-1 cells were transfected with 2 μg of TUC339 expressing vector or empty control vector using Jetprime (Polyplus-transfection SA, France) and, after 48 h, the cells were used for further experiments.
4.15. Genome-Wide Gene Expression Analysis
THP-1 differentiated cells were transfected with either si-TUC339, si-ctrl, TUC339 expression vector, or empty control vector for 48 h and were then treated with 1 μg/mL LPS for 16 h. The harvested cells were used for RNA isolation and microarray analysis by Kangchen Bio-tech Inc. (Shanghai, China) using Agilent human 4 × 44 k Gene Expression Microarray V2 (Agilent Technologies, Santa Clara, CA, USA).
4.16. Statistical Analysis
The data were expressed as the mean and standard error from at least three replicates. Comparisons between groups were performed using the two-tailed Student’s t test and results were considered to be statistically significant when p < 0.05.