Mol. Cells 2020; 43(9): 793-803
Published online August 31, 2020
https://doi.org/10.14348/molcells.2020.2307
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *zengx779@nenu.edu.cn
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
Myeloid-derived suppressor cells (MDSCs) promote tumour progression by contributing to angiogenesis, immunosuppression, and immunotherapy resistance. Although recent studies have shown that microRNAs (miRNAs) can promote the expansion of MDSCs in the tumour environment, the mechanisms involved in this process are largely unknown. Here, we report that microRNA 449c (miR-449c) expression was upregulated in myeloid progenitor cells upon activation of C-X-C motif chemokine receptor 2 (CXCR2) under tumour conditions. MiR-449c upregulation increased the generation of monocytic MDSCs (mo-MDSCs). The increased expression of miR-449c could target STAT6 mRNA in myeloid progenitor cells to shift the differentiation balance of myeloid progenitor cells and lead to an enhancement of the mo-MDSCs population in the tumour environment. Thus, our results demonstrate that the miR-449c/STAT6 axis is involved in the expansion of mo-MDSCs from myeloid progenitor cells upon activation of CXCR2, and thus, inhibition of miR-449c/STAT6 signalling may help to attenuate tumour progression.
Keywords C-X-C motif chemokine receptor 2, differentiation, microRNA 449c, mo-MDSCs, STAT6
Numerous studies have reported that myeloid-derived suppressor cells (MDSCs) accumulate in the bone marrow, spleen, and blood of patients with different types of cancer and various mouse tumour models (Hoechst et al., 2009; Ko et al., 2009; Ostrand-Rosenberg and Sinha, 2009). MDSCs play an important role in the suppression of T cell immune responses and contribute to tumour growth and metastasis (Wang et al., 2016; Ye et al., 2010). The accumulation of MDSCs under tumour conditions is mediated by various tumour-derived factors, such as interleukin (IL)-10, transforming growth factor-beta (TGF-β), IL-6, and prostaglandin E2 (PGE2) (Chomarat et al., 2000; Goddard et al., 2004; Halliday and Le, 2001; Jing et al., 2003). The increased expression and activity of these tumour-derived factors impairs the normal differentiation of myeloid progenitor cells, which results in the expansion of MDSCs (Gabrilovich et al., 2012).
MDSCs were identified as an immature myeloid heterogeneous population and include polymorphonuclear MDSCs (G-MDSCs), which have a granulocytic phenotype, and mononuclear MDSCs (mo-MDSCs), which have a monocytic phenotype (Movahedi et al., 2008). In mice, G-MDSCs express the Ly6G marker (CD45+CD11b+Ly6ClowLy6G+ cells), and mo-MDSCs express the Ly6C marker (CD45+CD11b+Ly6C+Ly6G– cells) (Gabrilovich et al., 2012). In humans, G-MDSCs are CD14–CD11b+CD33+CD15+ cells, and mo-MDSCs are CD14+HLA-DR- or CD11b+CD14–CD33+CD15– cells (Nagaraj and Gabrilovich, 2010). The recruitment of mo-MDSCs to the tumour site serves not only to inhibit the T cell immune response but also to reinforce and maintain a population of hypersuppressive G-MDSCs in the tumour microenvironment (Raber et al., 2014; Youn et al., 2013). However, the molecular mechanisms that regulate the accumulation of mo-MDSCs in tumour conditions remain to be elucidated. Understanding the molecular networks that regulate the expansion of mo-MDSCs is essential to identify potential therapeutic targets for cancer intervention.
MicroRNAs (miRNAs) are a class of endogenous short (approximately 20-25 nt) single-stranded RNAs that bind to the 3′ untranslated region (UTR) of target messenger RNA (mRNA), resulting in cleavage or translational repression (Bartel, 2004). The important role of miRNAs in regulating differentiation, apoptosis, cancer development, and metastasis has been well described (Majumder et al., 2015; Ren et al., 2015; Zang et al., 2015). Some miRNAs cooperate with transcription factors to regulate all aspects of haematopoiesis, including stem cell maintenance, lineage selection, cell expansion, and terminal differentiation (Kim et al., 2019). For instance, miR-129 and miR-520h regulate the differentiation of haematopoietic stem cells into more mature haematopoietic cells (Hong et al., 2015). During mouse lymphocyte differentiation and maturation, miR-150 and miR-146 have been shown to be upregulated (De Tullio et al., 2014; Saki et al., 2015). Some miRNAs have been reported to regulate the accumulation, activation, and immunomodulation of MDSCs in the tumour environment (Chen et al., 2015; El Gazzar, 2014; Wang et al., 2015). However, the regulation of miRNA expression in the tumour environment and the mechanisms by which tumour-derived miRNAs modulate mo-MDSCs expansion and activity remain to be determined.
Here, we profile miRNA expression in C-X-C motif chemokine receptor 2 (CXCR2)-transfected 32D clone 3 cells, which simulate granulocyte and macrophage progenitor cells (GMPs), and show upregulation of microRNA 449c (miR-449c) expression as a characteristic feature of myeloid progenitor cells in response to activation of CXCR2. Using pulldown experiments, we identified STAT6 mRNA as a target of miR-449c in myeloid progenitor cells during differentiation into mo-MDSCs in the tumour environment. Thus, our results demonstrate that the miR-449c/STAT6 axis is involved in CXCR2 activation-induced expansion of mo-MDSCs, suggesting that inhibition of miR-449c/STAT6 signalling may attenuate tumour progression.
B16F10 cells, HEK-293T cells, and 32D clone 3 cells were purchased from the American Type Culture Collection (ATCC). B16F10 cells and HEK-293T cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, USA) supplemented with 10% heat-inactivated foetal bovine serum (Corning, USA). 32D clone 3 cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated foetal bovine serum and 10% mouse IL-3 (213-13; PeproTech, USA). B16F10 cells, HEK-293T cells, and 32D clone 3 cells were all cultured with 1% penicillin/streptomycin.
C57BL/6J mice (female, 8-10 weeks old) used as wild-type (WT) controls were purchased from the Beijing Vital River Laboratory Animal Technology (China). CXCR2-deficient mice on a C57BL/6J background were provided by Dr. Hong Zhou (Department of Immunology, Nanjing Medical University). All mice were housed in a specific pathogen-free environment under protocols approved by the Animal Care Committee of Northeast Normal University, China (NENU/IACUC, AP20160928), and all experiments were performed under the Guidelines for the Care and Use of Laboratory Animals.
In total, 1 × 106 B16F10 cells were subcutaneously injected into the mice. The miR-449c inhibitor was injected into the tail vein of mice with transfection reagent (EntransterTM-in vivo; Engreen Biosystem, China) once a week from the tumour-bearing 1st week to 4th week. Then, the tumour size was measured twice every week from the tumour-bearing 2nd week to 4th week. The tumour volume was calculated using the following formula: a × b2/2, in which a represents the longest diameter and b represents the shortest diameter.
Total RNA of bone marrow cells or 32D clone 3 cells was extracted using TRIzol reagent (Invitrogen Life Technologies, USA) according to the manufacturer’s instructions. To determine miRNA expression, RNA was subsequently reverse-transcribed using the miRNA 1st Strand cDNA Synthesis Kit (by the stem-loop method) (Vazyme, China) according to the manufacturer’s protocol. The primer sequences that were used are listed in Supplementary Table S1. The expression levels of miR-449c and miR-6403 were quantified with SYBR Green Master Mix (Roche, Switzerland) using a miRNA-specific forward primer and a universal poly (T) adaptor reverse primer. The U6 small nuclear RNA was used as an internal reference. To determine mRNA expression, total RNA was reverse transcribed into cDNA according to the manufacturer’s instructions (Thermo Fisher Scientific, USA). Real-time PCR was performed on a Roche LightCycler 480 (Roche) real-time RT-PCR system. The primer sequences that were used are listed in Supplementary Table S2.
The STAT6 overexpression sequence was cloned into the pWPXLd vector (Addgene, USA), and the constructed plasmid or control plasmid was transfected into HEK-293T cells together with the packaging plasmid psPAX2 (Addgene) and the envelope plasmid pMD2.G (Addgene) using Lipofectamine 2000 reagent (Invitrogen Life Technologies). To overexpress STAT6 in tumour-bearing mice, the collected supernatant was concentrated and intrapleurally injected into two-week tumour-bearing mice four times every other day. To overexpress CXCR2 in 32D clone 3 cells, pEGFP-N1-CXCR2 was electroporated into the cells. The cells were collected by centrifugation for 5 min at 500
Transfection of mice bone marrow cells was performed with Lipofectamine 2000 (Invitrogen Life Technologies) according to the manufacturer's instructions. The cells were seeded into 24-well plates at 1 × 106/well. Mimics, inhibitors and negative control (RiboBio, China) of miR-449c were diluted at a working concentration of 20 nM. Mixed diluted transfection agent (Invitrogen Life Technologies) was mixed with diluted mimics, inhibitors or negative control of miR-449c, and then the mixture was incubated at room temperature for 10 min. Then, the transfection complexes were added to the cells, and the medium was changed to fresh complete medium 6 h post transfection. At 24 h post transfection, the cells were used for further experiments. The related sequences that were used are listed in Supplementary Table S4.
HEK-293T cells were seeded in 24-well plates overnight in the absence of antibiotics. The cells were then transfected with the STAT6 3′-UTR luciferase reporter plasmid, CEBPα 3′-UTR luciferase reporter plasmid or control vector (pGL3-Control) plus Renilla luciferase reporter plasmid (an internal control; Promega, USA) using Lipofectamine 2000 transfection reagent (Invitrogen Life Technologies). The treated cells were transfected with miR-449c mimic control or miR-449c mimics. To analyse the effects of the 3′-UTR on mRNA levels of target genes, firefly luciferase mRNA levels were measured by quantitative PCR (qPCR) and calibrated to that of Renilla. The related sequences that were used are listed in Supplementary Table S5.
Bone marrow cells were obtained from the femurs of control mice (Chatterjee et al., 2013). The tissues were ground and filtered through a 200 μm cell strainer, and erythrocytes were eliminated using hypotonic lysis buffer (155 mM NH4Cl, 0.1 mM EDTA, and 10 mM KHCO3). The remaining cells were transfected with miR-449c, then cultured in tumour-conditioned medium supplemented with GM-CSF (10 ng/ml, 315-03; PeproTech) for five days. CXCL1 (50 ng/ml, 250-11; PeproTech) and CXCL2 (50 ng/ml, 250-15; PeproTech) were added to the induction system.
Single-cell suspensions of the bone marrow, spleen, and blood samples were prepared and stained as previously described (Shi et al., 2018). The bone marrow and spleen were ground and filtered through a 200 μm cell strainer. To eliminate erythrocytes, a single-cell suspension was treated with hypotonic lysis buffer. The single-cell suspension was stained for 30 min at 4°C with appropriate dilutions of various combinations of the following fluorochrome-conjugated antibodies: anti-CD11b-FITC (clone M1/70), anti-CD45-PE/Cy7 (clone 30-F11), anti-Ly6G-APC/Cy7 (clone 1A8), anti-Ly6C-PE (clone AL-21), anti-CD115-PE/Cy7 (clone AFS98), and anti-CD115-APC (clone AFS98), which were all purchased from BD Biosciences (USA). The cells were further fixed using 10% formaldehyde (Sigma-Aldrich, USA) for 10 min, permeabilized using 0.1% Triton X-100 (Sigma-Aldrich) for 10 min, and then stained for Ki67 (clone 16A8, FITC-conjugated; BioLegend, USA). For the Annexin-V analysis, cells were stained according to the manufacturer’s instructions (BD Biosciences). The stained cells were acquired on FACSCanto II (BD Biosciences), and the data were analysed using FACSDiva software (BD Biosciences) and FlowJo 7.6.1 software (Treestar, USA). Dead cells and doublets were excluded based on the forward and side scatter.
Harvested cells were washed with phosphate-buffered saline (PBS), and protein was extracted using lysis buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.5], 1% NP-40, 1 mM EDTA, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM Na3VO4, 1 mM NaF, 1 mM PMSF and 0.1 mg/ml leupeptin/aprotinin) on ice for 30 min and centrifuged at 15,000
To isolate mo-MDSCs, tumour-bearing mice were sacrificed by tail vein injection of 4% EDTA. Blood was collected, and the erythrocytes were eliminated with hypotonic lysis buffer. The remaining cells were collected, and the mo-MDSCs were sorted with a Myeloid-Derived Suppressor Cell Isolation Kit using an AutoMACS sorter (Miltenyi Biotec, Germany) according to the manufacturer’s instructions. First, Gr-1high Ly6G+ cells were indirectly magnetically labelled with Anti-Ly6G-Biotin (MDSC-Kit) and Anti-Biotin MicroBeads. Then, the cell suspension was loaded onto a MACS column, which was placed in the magnetic field of a MACS Separator. The magnetically labelled Ly6G+ cells were retained in the column, and the unlabelled cells passed through. The unlabelled cells were depleted of Gr-1high Ly6G+ cells and pre-enriched for Gr-1dim Ly6G- myeloid cells. The Gr-1dim Ly6G- myeloid cells were separated according to Ly6C expression by Aria III (BD Biosciences).
For RNA sequencing (RNA-seq), 32D clone 3 cells transfected with CXCR2 or an empty vector were both incubated with CXCL1 and CXCL2 for 4 h and harvested. Cellular RNA was extracted using TRIzol reagent followed by a genomic DNA elimination step. RNA purity was assessed using the KaiaoK5500® Spectrophotometer (Kaiao, China). The RNA integrity and concentration were assessed using an RNA Nano 6000 Assay Kit with the Bioanalyzer 2100 system (Agilent Technologies, USA). Library construction and sequencing on an Illumina HiSeq 2500 instrument were performed at Annoroad Gene Technology Corporation (China). Bowtie2 v2.2.3 was used to build the genome index, and clean data were then aligned to the reference genome using HISAT2 v2.1.0. The level of gene expression was quantified using a software package called FPKM (fragments per kilobase of transcript per million mapped reads). DEGseq v1.18.0 was used for differential gene expression analysis between two samples with non-biological replicates. MiRNAs with a log2-fold change > 1 and a
All data analyses were performed using IBM SPSS (ver. 22; IBM, USA). Data are presented as the mean ± SD. Student’s
Previous studies have demonstrated that in GMPs, the expression of CXCR2 plays a critical role in the differentiation of haematopoietic progenitor cells into mo-MDSCs under tumour conditions (Han et al., 2019). To elucidate the potential role of non-coding RNA in CXCR2-mediated activation of myeloid progenitor cell differentiation, 32D clone 3 cells were transfected with CXCR2 to simulate GMPs (Supplementary Fig. S1) and examined using RNA sequencing. A log2-fold change > 3 and a
To investigate whether miR-449c is related to CXCR2-mediated differentiation of haematopoietic progenitor cells, bone marrow cells of WT or CXCR2–/– control mice were transfected with a miR-449c mimic (Supplementary Fig. S3) and cultured in the presence of CXCL1 and CXCL2. Overexpression of miR-449c significantly increased the percentage of mo-MDSCs in bone marrow cells of both WT and CXCR2–/– control mice. The transfection of the miR-449c mimic in bone marrow cells of CXCR2–/– control mice did not enhance the percentage of mo-MDSCs to the extent that was observed in the bone marrow cells of WT control mice (Figs. 2A and 2B). Overexpression of miR-449c had no effect on the suppressive activity of mo-MDSCs from WT compared to CXCR2–/– mice (Supplementary Fig. S4). These data suggest that miR-449c overexpression increased the accumulation of mo-MDSCs and that the increase was correlated with CXCR2 activation. The overexpression of miR-449c did not affect the proliferation and apoptosis of mo-MDSCs induced from WT or CXCR2–/– bone marrow cells (Figs. 2C and 2D). Upon appropriate stimulation, mo-MDSCs have been shown to differentiate into dendritic cells (CD11b+CD11c+) and macrophages (CD11b+F4/80+) (Poh et al., 2009), therefore, mo-MDSC differentiation was examined. The data showed that there were no significant differences in the percentage of dendritic cells or macrophages derived from mo-MDSCs sorting from WT compared to CXCR2–/– tumour-bearing mice (Supplementary Fig. S5). Next, mo-MDSCs were sorted from the blood of WT or CXCR2–/– tumour-bearing mice, miR-449c was overexpressed in these cells, and the percentage of dendritic cells or macrophages derived from mo-MDSCs was examined. The results showed that miR-449c overexpression did not influence the ability of mo-MDSCs to differentiate into dendritic cells or macrophages (Figs. 2E and 2F). Taken together, these results indicate that miR-449c overexpression promotes the differentiation of myeloid progenitor cells to mo-MDSCs under tumour conditions.
To determine whether increased mo-MDSCs generation resulting from upregulation of miR-449c under tumour conditions contributes to tumour progression, a miR-449c inhibitor was injected into WT tumour-bearing mice via the tail vein once a week during tumour-bearing weeks one through four. The results showed that miR-449 expression was inhibited in bone marrow cells (Fig. 3A), and the miR-449c inhibitor decreased the percentage of mo-MDSCs in the bone marrow and blood of tumour-bearing mice (Figs. 3B and 3C). Furthermore, a significant inhibition of melanoma growth was observed in miR-449c inhibitor-injected mice compared with untreated mice after 2 weeks of tumour bearing (Fig. 3D). These results suggest that miR-449c promotes tumour progression by increasing the accumulation of mo-MDSCs.
Our data thus far demonstrate that miR-449c expression is increased in bone marrow cells upon CXCR2 activation, but the molecular mechanisms underlying these functions require further exploration. Some transcription factors, such as CEBPα, CEBPβ, and STAT6, are important for the differentiation of myeloid progenitor cells (Friedman, 2015; Marigo et al., 2010; Munera et al., 2010); therefore, we hypothesized that miR-449c promotes the accumulation of mo-MDSCs by targeting these transcription factors. To test this hypothesis, miR-449c was overexpressed in bone marrow cells. Overexpression of miR-449c resulted in a decrease in CEBPα, CEBPβ, and STAT6 mRNA expression in bone marrow cells (Fig. 4A). CEBPα and STAT6 protein expression was decreased in bone marrow cells with miR-449c overexpression and increased upon inhibition of miR-449c expression (Figs. 4B and 4C). In contrast, CEBPβ protein expression in bone marrow cells was not dramatically changed upon increasing or inhibiting miR-449c expression (Figs. 4B and 4C). These results suggest that miR-449c may target CEBPα and STAT6 to regulate the accumulation of mo-MDSCs. An online bioinformatics database (TargetScan; http://www.targetscan.org/vert_71/) was used to identify putative targets of miR-449c in bone marrow cells. Based on the predictions from TargetScan, the 3′-UTR of the STAT6 gene was revealed to contain the binding sequences of miR-449c, whereas the 3′-UTR of the CEBPα gene was not predicted to contain the binding sequences, suggesting that STAT6 may be a downstream target of miR-449c (Fig. 4D). To determine whether miR-449c can interact with the STAT6 3′-UTR or CEBPα 3′-UTR, luciferase assays were performed. Overexpression of miR-449c in bone marrow cells significantly reduced luciferase mRNA levels of the STAT6 3′- UTR but had no effect on that of the control vector or CEBPα 3′-UTR (Fig. 4E). The modulation of STAT6 expression and STAT6-targeted gene expression (
Immunoblot analyses showed that the expression of STAT6 was increased in bone marrow cells of tumour-bearing mice, and this increase was further enhanced in mice after intrapleural injection with a lentivirus-based STAT6 overexpression sequence (Fig. 5A). The percentage of mo-MDSCs and G-MDSCs was analysed, and the results showed that the percentage of mo-MDSCs was decreased in the bone marrow, spleen, and blood of the tumour-bearing mice with STAT6 overexpression (Fig. 5B, Supplementary Fig. S7). The percentage of G-MDSCs was increased in the bone marrow and blood but was decreased in the spleen of tumour-bearing mice overexpressing STAT6 (Fig. 5C, Supplementary Fig. S7). To further confirm whether STAT6 is the mediator of the biological function of miR-449c in bone marrow cells, cells from WT control mice were transfected with STAT6, the miR-449c mimic and STAT6, or the miR-449c mimic control and STAT6. The results of immunoblot analyses showed that miR-449c overexpression inhibited STAT6 expression (Fig. 5D). The percentage of mo-MDSCs and G-MDSCs that were derived from the transfected cells was examined. The results showed that STAT6 overexpression decreased the percentage of mo-MDSCs and increased the percentage of G-MDSCs. In contrast, the opposite results were found in the group of cells transfected with the miR-449c mimic and STAT6. This was because the expression of STAT6 was inhibited by miR-449c overexpression (Figs. 5E and 5F, Supplementary Fig. S8). These data suggest that overexpression of miR-449c decreases the inhibitory effect of STAT6 on the generation of mo-MDSCs.
Several studies have identified miR449c as a novel cancer-related miRNA and an important regulator of proliferation and invasion in nonsmall cell lung cancer and gastric cancer (Chen et al., 2018; Miao et al., 2013). In liver cancer, miR449c acts as a tumour suppressor by promoting cell death and inhibiting cell migration (Sandbothe et al., 2017). Furthermore, miR449c belongs to the family of miRNAs that plays an essential role in regulating brain development, motile ciliogenesis, and spermatogenesis (Wu et al., 2014). The present study demonstrated that miR-449c was upregulated in myeloid progenitor cells of WT tumour-bearing mice compared with CXCR2–/– tumour-bearing mice (Fig. 1F), suggesting that miR-449c expression was dependent upon activation of CXCR2 under tumour conditions. CXCR2 activation-mediated expression of miR-449c was found to be highest in bone marrow cells at 2 weeks of tumour-bearing (Fig. 1D), and the percentage of mo-MDSCs in the bone marrow was increased in tumour-bearing mice at 3 weeks (Han et al., 2019). These data indicate that increased miR-449c expression may be a necessary event for the expansion of mo-MDSCs.
It has been reported that miR-449c is downregulated in several tumour tissues (Chen et al., 2018; Miao et al., 2013), but the mechanism underlying this downregulation remains largely unknown. The results presented here demonstrate that the upregulated expression of miR-449c in bone marrow is a result of CXCR2 activation in myeloid progenitor cells, suggesting that miR-449c may play a cell type-specific role across different tissues.
We also reported a novel discovery that STAT6 is a downstream target of miR-449c in myeloid progenitor cells. We showed that STAT6 promotes the expansion of G-MDSCs, but weakens the expansion of mo-MDSCs in a subcutaneous model of melanoma (Figs. 5B and 5C). In contrast, STAT6 has been previously reported to promote the expansion of G-MDSCs and mo-MDSCs in a colorectal cancer model (Jayakumar and Bothwell, 2017; Leon-Cabrera et al., 2017). The different effects of STAT6 on the expansion of mo-MDSCs may be caused by the differences in tumour heterogeneity. Moreover, we increased the expression of STAT6 in mice by intrapleural injection with a lentivirus-based STAT6 overexpression sequence. Knocking down STAT6 may have a wider impact than overexpressing STAT6 in mice. MiR-449c overexpression blocks the inhibitory effect of STAT6 on the expansion of mo-MDSCs (Figs. 5E and 5F). These data suggest that miR-449c may weaken the STAT6-mediated inhibition of the differentiation of myeloid progenitor cell to mo-MDSCs under tumour conditions. It is important to note that the STAT6 pathway was originally reported to regulate the survival and accumulation of MDSCs via CD124 (Roth et al., 2012), and was also shown to mediate MDSC suppression by increasing the activity of arginase-1 (Condamine et al., 2015). Thus, our study serves to further understand the role of the STAT6 pathway in the accumulation of MDSCs. A previous study showed that CXCR2 regulates the differentiation of GMPs into mo-MDSCs through the ERK/STAT3 pathway (Han et al., 2019). Therefore, our results suggest that the STAT family of transcription factors performs a different function in the accumulation of mo-MDSCs and G-MDSCs.
In summary, the present study demonstrated that miR-449c expression was upregulated in myeloid progenitor cells in response to CXCR2 activation. The increased expression of miR449c was associated with the generation of mo-MDSCs. Overexpression of miR449c contributed to the expansion of mo-MDSCs, whereas knockdown of miR449c expression inhibited differentiation of myeloid progenitor cells to mo-MDSCs. Finally, STAT6 was identified as a novel downstream target and functional mediator of miR449c during the differentiation of myeloid progenitor cells (Fig. 6).
We thank Dr. Hong Zhou (Department of Immunology, Nanjing Medical University) for the support with C57BL/6J CXCR2–/– mice. This work was funded by The National Nature Science Foundation of China. The grant numbers are 81670095, 31870888, and 81372288.
X.H. and T.L. performed experiments. X.H., T.L., Y.S., W.Y., and D.W. analyzed the data. X.Z. supervised the research. X.H. and X.Z. wrote the manuscript. All authors read and approved the manuscript.
The authors have no potential conflicts of interest to disclose.
Mol. Cells 2020; 43(9): 793-803
Published online September 30, 2020 https://doi.org/10.14348/molcells.2020.2307
Copyright © The Korean Society for Molecular and Cellular Biology.
Xiaoqing Han , Tao Luan
, Yingying Sun
, Wenyi Yan
, Dake Wang
, and Xianlu Zeng*
The Key Laboratory of Molecular Epigenetics of Ministry of Education, Institute of Genetics and Cytology, School of Life Sciences, Northeast Normal University, Changchun 130024, China
Correspondence to:*zengx779@nenu.edu.cn
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
Myeloid-derived suppressor cells (MDSCs) promote tumour progression by contributing to angiogenesis, immunosuppression, and immunotherapy resistance. Although recent studies have shown that microRNAs (miRNAs) can promote the expansion of MDSCs in the tumour environment, the mechanisms involved in this process are largely unknown. Here, we report that microRNA 449c (miR-449c) expression was upregulated in myeloid progenitor cells upon activation of C-X-C motif chemokine receptor 2 (CXCR2) under tumour conditions. MiR-449c upregulation increased the generation of monocytic MDSCs (mo-MDSCs). The increased expression of miR-449c could target STAT6 mRNA in myeloid progenitor cells to shift the differentiation balance of myeloid progenitor cells and lead to an enhancement of the mo-MDSCs population in the tumour environment. Thus, our results demonstrate that the miR-449c/STAT6 axis is involved in the expansion of mo-MDSCs from myeloid progenitor cells upon activation of CXCR2, and thus, inhibition of miR-449c/STAT6 signalling may help to attenuate tumour progression.
Keywords: C-X-C motif chemokine receptor 2, differentiation, microRNA 449c, mo-MDSCs, STAT6
Numerous studies have reported that myeloid-derived suppressor cells (MDSCs) accumulate in the bone marrow, spleen, and blood of patients with different types of cancer and various mouse tumour models (Hoechst et al., 2009; Ko et al., 2009; Ostrand-Rosenberg and Sinha, 2009). MDSCs play an important role in the suppression of T cell immune responses and contribute to tumour growth and metastasis (Wang et al., 2016; Ye et al., 2010). The accumulation of MDSCs under tumour conditions is mediated by various tumour-derived factors, such as interleukin (IL)-10, transforming growth factor-beta (TGF-β), IL-6, and prostaglandin E2 (PGE2) (Chomarat et al., 2000; Goddard et al., 2004; Halliday and Le, 2001; Jing et al., 2003). The increased expression and activity of these tumour-derived factors impairs the normal differentiation of myeloid progenitor cells, which results in the expansion of MDSCs (Gabrilovich et al., 2012).
MDSCs were identified as an immature myeloid heterogeneous population and include polymorphonuclear MDSCs (G-MDSCs), which have a granulocytic phenotype, and mononuclear MDSCs (mo-MDSCs), which have a monocytic phenotype (Movahedi et al., 2008). In mice, G-MDSCs express the Ly6G marker (CD45+CD11b+Ly6ClowLy6G+ cells), and mo-MDSCs express the Ly6C marker (CD45+CD11b+Ly6C+Ly6G– cells) (Gabrilovich et al., 2012). In humans, G-MDSCs are CD14–CD11b+CD33+CD15+ cells, and mo-MDSCs are CD14+HLA-DR- or CD11b+CD14–CD33+CD15– cells (Nagaraj and Gabrilovich, 2010). The recruitment of mo-MDSCs to the tumour site serves not only to inhibit the T cell immune response but also to reinforce and maintain a population of hypersuppressive G-MDSCs in the tumour microenvironment (Raber et al., 2014; Youn et al., 2013). However, the molecular mechanisms that regulate the accumulation of mo-MDSCs in tumour conditions remain to be elucidated. Understanding the molecular networks that regulate the expansion of mo-MDSCs is essential to identify potential therapeutic targets for cancer intervention.
MicroRNAs (miRNAs) are a class of endogenous short (approximately 20-25 nt) single-stranded RNAs that bind to the 3′ untranslated region (UTR) of target messenger RNA (mRNA), resulting in cleavage or translational repression (Bartel, 2004). The important role of miRNAs in regulating differentiation, apoptosis, cancer development, and metastasis has been well described (Majumder et al., 2015; Ren et al., 2015; Zang et al., 2015). Some miRNAs cooperate with transcription factors to regulate all aspects of haematopoiesis, including stem cell maintenance, lineage selection, cell expansion, and terminal differentiation (Kim et al., 2019). For instance, miR-129 and miR-520h regulate the differentiation of haematopoietic stem cells into more mature haematopoietic cells (Hong et al., 2015). During mouse lymphocyte differentiation and maturation, miR-150 and miR-146 have been shown to be upregulated (De Tullio et al., 2014; Saki et al., 2015). Some miRNAs have been reported to regulate the accumulation, activation, and immunomodulation of MDSCs in the tumour environment (Chen et al., 2015; El Gazzar, 2014; Wang et al., 2015). However, the regulation of miRNA expression in the tumour environment and the mechanisms by which tumour-derived miRNAs modulate mo-MDSCs expansion and activity remain to be determined.
Here, we profile miRNA expression in C-X-C motif chemokine receptor 2 (CXCR2)-transfected 32D clone 3 cells, which simulate granulocyte and macrophage progenitor cells (GMPs), and show upregulation of microRNA 449c (miR-449c) expression as a characteristic feature of myeloid progenitor cells in response to activation of CXCR2. Using pulldown experiments, we identified STAT6 mRNA as a target of miR-449c in myeloid progenitor cells during differentiation into mo-MDSCs in the tumour environment. Thus, our results demonstrate that the miR-449c/STAT6 axis is involved in CXCR2 activation-induced expansion of mo-MDSCs, suggesting that inhibition of miR-449c/STAT6 signalling may attenuate tumour progression.
B16F10 cells, HEK-293T cells, and 32D clone 3 cells were purchased from the American Type Culture Collection (ATCC). B16F10 cells and HEK-293T cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, USA) supplemented with 10% heat-inactivated foetal bovine serum (Corning, USA). 32D clone 3 cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated foetal bovine serum and 10% mouse IL-3 (213-13; PeproTech, USA). B16F10 cells, HEK-293T cells, and 32D clone 3 cells were all cultured with 1% penicillin/streptomycin.
C57BL/6J mice (female, 8-10 weeks old) used as wild-type (WT) controls were purchased from the Beijing Vital River Laboratory Animal Technology (China). CXCR2-deficient mice on a C57BL/6J background were provided by Dr. Hong Zhou (Department of Immunology, Nanjing Medical University). All mice were housed in a specific pathogen-free environment under protocols approved by the Animal Care Committee of Northeast Normal University, China (NENU/IACUC, AP20160928), and all experiments were performed under the Guidelines for the Care and Use of Laboratory Animals.
In total, 1 × 106 B16F10 cells were subcutaneously injected into the mice. The miR-449c inhibitor was injected into the tail vein of mice with transfection reagent (EntransterTM-in vivo; Engreen Biosystem, China) once a week from the tumour-bearing 1st week to 4th week. Then, the tumour size was measured twice every week from the tumour-bearing 2nd week to 4th week. The tumour volume was calculated using the following formula: a × b2/2, in which a represents the longest diameter and b represents the shortest diameter.
Total RNA of bone marrow cells or 32D clone 3 cells was extracted using TRIzol reagent (Invitrogen Life Technologies, USA) according to the manufacturer’s instructions. To determine miRNA expression, RNA was subsequently reverse-transcribed using the miRNA 1st Strand cDNA Synthesis Kit (by the stem-loop method) (Vazyme, China) according to the manufacturer’s protocol. The primer sequences that were used are listed in Supplementary Table S1. The expression levels of miR-449c and miR-6403 were quantified with SYBR Green Master Mix (Roche, Switzerland) using a miRNA-specific forward primer and a universal poly (T) adaptor reverse primer. The U6 small nuclear RNA was used as an internal reference. To determine mRNA expression, total RNA was reverse transcribed into cDNA according to the manufacturer’s instructions (Thermo Fisher Scientific, USA). Real-time PCR was performed on a Roche LightCycler 480 (Roche) real-time RT-PCR system. The primer sequences that were used are listed in Supplementary Table S2.
The STAT6 overexpression sequence was cloned into the pWPXLd vector (Addgene, USA), and the constructed plasmid or control plasmid was transfected into HEK-293T cells together with the packaging plasmid psPAX2 (Addgene) and the envelope plasmid pMD2.G (Addgene) using Lipofectamine 2000 reagent (Invitrogen Life Technologies). To overexpress STAT6 in tumour-bearing mice, the collected supernatant was concentrated and intrapleurally injected into two-week tumour-bearing mice four times every other day. To overexpress CXCR2 in 32D clone 3 cells, pEGFP-N1-CXCR2 was electroporated into the cells. The cells were collected by centrifugation for 5 min at 500
Transfection of mice bone marrow cells was performed with Lipofectamine 2000 (Invitrogen Life Technologies) according to the manufacturer's instructions. The cells were seeded into 24-well plates at 1 × 106/well. Mimics, inhibitors and negative control (RiboBio, China) of miR-449c were diluted at a working concentration of 20 nM. Mixed diluted transfection agent (Invitrogen Life Technologies) was mixed with diluted mimics, inhibitors or negative control of miR-449c, and then the mixture was incubated at room temperature for 10 min. Then, the transfection complexes were added to the cells, and the medium was changed to fresh complete medium 6 h post transfection. At 24 h post transfection, the cells were used for further experiments. The related sequences that were used are listed in Supplementary Table S4.
HEK-293T cells were seeded in 24-well plates overnight in the absence of antibiotics. The cells were then transfected with the STAT6 3′-UTR luciferase reporter plasmid, CEBPα 3′-UTR luciferase reporter plasmid or control vector (pGL3-Control) plus Renilla luciferase reporter plasmid (an internal control; Promega, USA) using Lipofectamine 2000 transfection reagent (Invitrogen Life Technologies). The treated cells were transfected with miR-449c mimic control or miR-449c mimics. To analyse the effects of the 3′-UTR on mRNA levels of target genes, firefly luciferase mRNA levels were measured by quantitative PCR (qPCR) and calibrated to that of Renilla. The related sequences that were used are listed in Supplementary Table S5.
Bone marrow cells were obtained from the femurs of control mice (Chatterjee et al., 2013). The tissues were ground and filtered through a 200 μm cell strainer, and erythrocytes were eliminated using hypotonic lysis buffer (155 mM NH4Cl, 0.1 mM EDTA, and 10 mM KHCO3). The remaining cells were transfected with miR-449c, then cultured in tumour-conditioned medium supplemented with GM-CSF (10 ng/ml, 315-03; PeproTech) for five days. CXCL1 (50 ng/ml, 250-11; PeproTech) and CXCL2 (50 ng/ml, 250-15; PeproTech) were added to the induction system.
Single-cell suspensions of the bone marrow, spleen, and blood samples were prepared and stained as previously described (Shi et al., 2018). The bone marrow and spleen were ground and filtered through a 200 μm cell strainer. To eliminate erythrocytes, a single-cell suspension was treated with hypotonic lysis buffer. The single-cell suspension was stained for 30 min at 4°C with appropriate dilutions of various combinations of the following fluorochrome-conjugated antibodies: anti-CD11b-FITC (clone M1/70), anti-CD45-PE/Cy7 (clone 30-F11), anti-Ly6G-APC/Cy7 (clone 1A8), anti-Ly6C-PE (clone AL-21), anti-CD115-PE/Cy7 (clone AFS98), and anti-CD115-APC (clone AFS98), which were all purchased from BD Biosciences (USA). The cells were further fixed using 10% formaldehyde (Sigma-Aldrich, USA) for 10 min, permeabilized using 0.1% Triton X-100 (Sigma-Aldrich) for 10 min, and then stained for Ki67 (clone 16A8, FITC-conjugated; BioLegend, USA). For the Annexin-V analysis, cells were stained according to the manufacturer’s instructions (BD Biosciences). The stained cells were acquired on FACSCanto II (BD Biosciences), and the data were analysed using FACSDiva software (BD Biosciences) and FlowJo 7.6.1 software (Treestar, USA). Dead cells and doublets were excluded based on the forward and side scatter.
Harvested cells were washed with phosphate-buffered saline (PBS), and protein was extracted using lysis buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.5], 1% NP-40, 1 mM EDTA, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM Na3VO4, 1 mM NaF, 1 mM PMSF and 0.1 mg/ml leupeptin/aprotinin) on ice for 30 min and centrifuged at 15,000
To isolate mo-MDSCs, tumour-bearing mice were sacrificed by tail vein injection of 4% EDTA. Blood was collected, and the erythrocytes were eliminated with hypotonic lysis buffer. The remaining cells were collected, and the mo-MDSCs were sorted with a Myeloid-Derived Suppressor Cell Isolation Kit using an AutoMACS sorter (Miltenyi Biotec, Germany) according to the manufacturer’s instructions. First, Gr-1high Ly6G+ cells were indirectly magnetically labelled with Anti-Ly6G-Biotin (MDSC-Kit) and Anti-Biotin MicroBeads. Then, the cell suspension was loaded onto a MACS column, which was placed in the magnetic field of a MACS Separator. The magnetically labelled Ly6G+ cells were retained in the column, and the unlabelled cells passed through. The unlabelled cells were depleted of Gr-1high Ly6G+ cells and pre-enriched for Gr-1dim Ly6G- myeloid cells. The Gr-1dim Ly6G- myeloid cells were separated according to Ly6C expression by Aria III (BD Biosciences).
For RNA sequencing (RNA-seq), 32D clone 3 cells transfected with CXCR2 or an empty vector were both incubated with CXCL1 and CXCL2 for 4 h and harvested. Cellular RNA was extracted using TRIzol reagent followed by a genomic DNA elimination step. RNA purity was assessed using the KaiaoK5500® Spectrophotometer (Kaiao, China). The RNA integrity and concentration were assessed using an RNA Nano 6000 Assay Kit with the Bioanalyzer 2100 system (Agilent Technologies, USA). Library construction and sequencing on an Illumina HiSeq 2500 instrument were performed at Annoroad Gene Technology Corporation (China). Bowtie2 v2.2.3 was used to build the genome index, and clean data were then aligned to the reference genome using HISAT2 v2.1.0. The level of gene expression was quantified using a software package called FPKM (fragments per kilobase of transcript per million mapped reads). DEGseq v1.18.0 was used for differential gene expression analysis between two samples with non-biological replicates. MiRNAs with a log2-fold change > 1 and a
All data analyses were performed using IBM SPSS (ver. 22; IBM, USA). Data are presented as the mean ± SD. Student’s
Previous studies have demonstrated that in GMPs, the expression of CXCR2 plays a critical role in the differentiation of haematopoietic progenitor cells into mo-MDSCs under tumour conditions (Han et al., 2019). To elucidate the potential role of non-coding RNA in CXCR2-mediated activation of myeloid progenitor cell differentiation, 32D clone 3 cells were transfected with CXCR2 to simulate GMPs (Supplementary Fig. S1) and examined using RNA sequencing. A log2-fold change > 3 and a
To investigate whether miR-449c is related to CXCR2-mediated differentiation of haematopoietic progenitor cells, bone marrow cells of WT or CXCR2–/– control mice were transfected with a miR-449c mimic (Supplementary Fig. S3) and cultured in the presence of CXCL1 and CXCL2. Overexpression of miR-449c significantly increased the percentage of mo-MDSCs in bone marrow cells of both WT and CXCR2–/– control mice. The transfection of the miR-449c mimic in bone marrow cells of CXCR2–/– control mice did not enhance the percentage of mo-MDSCs to the extent that was observed in the bone marrow cells of WT control mice (Figs. 2A and 2B). Overexpression of miR-449c had no effect on the suppressive activity of mo-MDSCs from WT compared to CXCR2–/– mice (Supplementary Fig. S4). These data suggest that miR-449c overexpression increased the accumulation of mo-MDSCs and that the increase was correlated with CXCR2 activation. The overexpression of miR-449c did not affect the proliferation and apoptosis of mo-MDSCs induced from WT or CXCR2–/– bone marrow cells (Figs. 2C and 2D). Upon appropriate stimulation, mo-MDSCs have been shown to differentiate into dendritic cells (CD11b+CD11c+) and macrophages (CD11b+F4/80+) (Poh et al., 2009), therefore, mo-MDSC differentiation was examined. The data showed that there were no significant differences in the percentage of dendritic cells or macrophages derived from mo-MDSCs sorting from WT compared to CXCR2–/– tumour-bearing mice (Supplementary Fig. S5). Next, mo-MDSCs were sorted from the blood of WT or CXCR2–/– tumour-bearing mice, miR-449c was overexpressed in these cells, and the percentage of dendritic cells or macrophages derived from mo-MDSCs was examined. The results showed that miR-449c overexpression did not influence the ability of mo-MDSCs to differentiate into dendritic cells or macrophages (Figs. 2E and 2F). Taken together, these results indicate that miR-449c overexpression promotes the differentiation of myeloid progenitor cells to mo-MDSCs under tumour conditions.
To determine whether increased mo-MDSCs generation resulting from upregulation of miR-449c under tumour conditions contributes to tumour progression, a miR-449c inhibitor was injected into WT tumour-bearing mice via the tail vein once a week during tumour-bearing weeks one through four. The results showed that miR-449 expression was inhibited in bone marrow cells (Fig. 3A), and the miR-449c inhibitor decreased the percentage of mo-MDSCs in the bone marrow and blood of tumour-bearing mice (Figs. 3B and 3C). Furthermore, a significant inhibition of melanoma growth was observed in miR-449c inhibitor-injected mice compared with untreated mice after 2 weeks of tumour bearing (Fig. 3D). These results suggest that miR-449c promotes tumour progression by increasing the accumulation of mo-MDSCs.
Our data thus far demonstrate that miR-449c expression is increased in bone marrow cells upon CXCR2 activation, but the molecular mechanisms underlying these functions require further exploration. Some transcription factors, such as CEBPα, CEBPβ, and STAT6, are important for the differentiation of myeloid progenitor cells (Friedman, 2015; Marigo et al., 2010; Munera et al., 2010); therefore, we hypothesized that miR-449c promotes the accumulation of mo-MDSCs by targeting these transcription factors. To test this hypothesis, miR-449c was overexpressed in bone marrow cells. Overexpression of miR-449c resulted in a decrease in CEBPα, CEBPβ, and STAT6 mRNA expression in bone marrow cells (Fig. 4A). CEBPα and STAT6 protein expression was decreased in bone marrow cells with miR-449c overexpression and increased upon inhibition of miR-449c expression (Figs. 4B and 4C). In contrast, CEBPβ protein expression in bone marrow cells was not dramatically changed upon increasing or inhibiting miR-449c expression (Figs. 4B and 4C). These results suggest that miR-449c may target CEBPα and STAT6 to regulate the accumulation of mo-MDSCs. An online bioinformatics database (TargetScan; http://www.targetscan.org/vert_71/) was used to identify putative targets of miR-449c in bone marrow cells. Based on the predictions from TargetScan, the 3′-UTR of the STAT6 gene was revealed to contain the binding sequences of miR-449c, whereas the 3′-UTR of the CEBPα gene was not predicted to contain the binding sequences, suggesting that STAT6 may be a downstream target of miR-449c (Fig. 4D). To determine whether miR-449c can interact with the STAT6 3′-UTR or CEBPα 3′-UTR, luciferase assays were performed. Overexpression of miR-449c in bone marrow cells significantly reduced luciferase mRNA levels of the STAT6 3′- UTR but had no effect on that of the control vector or CEBPα 3′-UTR (Fig. 4E). The modulation of STAT6 expression and STAT6-targeted gene expression (
Immunoblot analyses showed that the expression of STAT6 was increased in bone marrow cells of tumour-bearing mice, and this increase was further enhanced in mice after intrapleural injection with a lentivirus-based STAT6 overexpression sequence (Fig. 5A). The percentage of mo-MDSCs and G-MDSCs was analysed, and the results showed that the percentage of mo-MDSCs was decreased in the bone marrow, spleen, and blood of the tumour-bearing mice with STAT6 overexpression (Fig. 5B, Supplementary Fig. S7). The percentage of G-MDSCs was increased in the bone marrow and blood but was decreased in the spleen of tumour-bearing mice overexpressing STAT6 (Fig. 5C, Supplementary Fig. S7). To further confirm whether STAT6 is the mediator of the biological function of miR-449c in bone marrow cells, cells from WT control mice were transfected with STAT6, the miR-449c mimic and STAT6, or the miR-449c mimic control and STAT6. The results of immunoblot analyses showed that miR-449c overexpression inhibited STAT6 expression (Fig. 5D). The percentage of mo-MDSCs and G-MDSCs that were derived from the transfected cells was examined. The results showed that STAT6 overexpression decreased the percentage of mo-MDSCs and increased the percentage of G-MDSCs. In contrast, the opposite results were found in the group of cells transfected with the miR-449c mimic and STAT6. This was because the expression of STAT6 was inhibited by miR-449c overexpression (Figs. 5E and 5F, Supplementary Fig. S8). These data suggest that overexpression of miR-449c decreases the inhibitory effect of STAT6 on the generation of mo-MDSCs.
Several studies have identified miR449c as a novel cancer-related miRNA and an important regulator of proliferation and invasion in nonsmall cell lung cancer and gastric cancer (Chen et al., 2018; Miao et al., 2013). In liver cancer, miR449c acts as a tumour suppressor by promoting cell death and inhibiting cell migration (Sandbothe et al., 2017). Furthermore, miR449c belongs to the family of miRNAs that plays an essential role in regulating brain development, motile ciliogenesis, and spermatogenesis (Wu et al., 2014). The present study demonstrated that miR-449c was upregulated in myeloid progenitor cells of WT tumour-bearing mice compared with CXCR2–/– tumour-bearing mice (Fig. 1F), suggesting that miR-449c expression was dependent upon activation of CXCR2 under tumour conditions. CXCR2 activation-mediated expression of miR-449c was found to be highest in bone marrow cells at 2 weeks of tumour-bearing (Fig. 1D), and the percentage of mo-MDSCs in the bone marrow was increased in tumour-bearing mice at 3 weeks (Han et al., 2019). These data indicate that increased miR-449c expression may be a necessary event for the expansion of mo-MDSCs.
It has been reported that miR-449c is downregulated in several tumour tissues (Chen et al., 2018; Miao et al., 2013), but the mechanism underlying this downregulation remains largely unknown. The results presented here demonstrate that the upregulated expression of miR-449c in bone marrow is a result of CXCR2 activation in myeloid progenitor cells, suggesting that miR-449c may play a cell type-specific role across different tissues.
We also reported a novel discovery that STAT6 is a downstream target of miR-449c in myeloid progenitor cells. We showed that STAT6 promotes the expansion of G-MDSCs, but weakens the expansion of mo-MDSCs in a subcutaneous model of melanoma (Figs. 5B and 5C). In contrast, STAT6 has been previously reported to promote the expansion of G-MDSCs and mo-MDSCs in a colorectal cancer model (Jayakumar and Bothwell, 2017; Leon-Cabrera et al., 2017). The different effects of STAT6 on the expansion of mo-MDSCs may be caused by the differences in tumour heterogeneity. Moreover, we increased the expression of STAT6 in mice by intrapleural injection with a lentivirus-based STAT6 overexpression sequence. Knocking down STAT6 may have a wider impact than overexpressing STAT6 in mice. MiR-449c overexpression blocks the inhibitory effect of STAT6 on the expansion of mo-MDSCs (Figs. 5E and 5F). These data suggest that miR-449c may weaken the STAT6-mediated inhibition of the differentiation of myeloid progenitor cell to mo-MDSCs under tumour conditions. It is important to note that the STAT6 pathway was originally reported to regulate the survival and accumulation of MDSCs via CD124 (Roth et al., 2012), and was also shown to mediate MDSC suppression by increasing the activity of arginase-1 (Condamine et al., 2015). Thus, our study serves to further understand the role of the STAT6 pathway in the accumulation of MDSCs. A previous study showed that CXCR2 regulates the differentiation of GMPs into mo-MDSCs through the ERK/STAT3 pathway (Han et al., 2019). Therefore, our results suggest that the STAT family of transcription factors performs a different function in the accumulation of mo-MDSCs and G-MDSCs.
In summary, the present study demonstrated that miR-449c expression was upregulated in myeloid progenitor cells in response to CXCR2 activation. The increased expression of miR449c was associated with the generation of mo-MDSCs. Overexpression of miR449c contributed to the expansion of mo-MDSCs, whereas knockdown of miR449c expression inhibited differentiation of myeloid progenitor cells to mo-MDSCs. Finally, STAT6 was identified as a novel downstream target and functional mediator of miR449c during the differentiation of myeloid progenitor cells (Fig. 6).
We thank Dr. Hong Zhou (Department of Immunology, Nanjing Medical University) for the support with C57BL/6J CXCR2–/– mice. This work was funded by The National Nature Science Foundation of China. The grant numbers are 81670095, 31870888, and 81372288.
X.H. and T.L. performed experiments. X.H., T.L., Y.S., W.Y., and D.W. analyzed the data. X.Z. supervised the research. X.H. and X.Z. wrote the manuscript. All authors read and approved the manuscript.
The authors have no potential conflicts of interest to disclose.
Jin Eom, Juhyun Choi, Sung-Suk Suh, and Jong Bae Seo
Mol. Cells 2022; 45(12): 963-975 https://doi.org/10.14348/molcells.2022.0123Jianming Hua, Ning Shen, Jingkai Wang, Yiqing Tao, Fangcai Li, Qixin Chen, and Xiaopeng Zhou
Mol. Cells 2019; 42(9): 661-671 https://doi.org/10.14348/molcells.2019.0098Yujeong Oh, and Jiwon Jang
Mol. Cells 2019; 42(3): 200-209 https://doi.org/10.14348/molcells.2019.2439