the pseudogene olfr29-ps1 promotes the suppressive ... · a lncrna pseudogene,...

Research Article

The Pseudogene Olfr29-ps1 Promotes theSuppressive Function and Differentiation ofMonocytic MDSCsWencong Shang1,2,3, Yunhuan Gao1,2,3, Zhenzhen Tang1,2,3, Yuan Zhang1,2,3, andRongcun Yang1,2,3

Abstract

Long noncoding RNA (lncRNA) plays a critical role inmany biological processes, such as cell differentiation anddevelopment. However, few studies about lncRNAs regulat-ing the differentiation and development of myeloid-derivedsuppressor cells (MDSCs) exist. In this study, we identifieda lncRNA pseudogene, Olfr29-ps1, which was expressedin MDSCs and upregulated by the proinflammatory cyto-kine IL6. The Olfr29-ps1 in vertebrates is conserved, andthe similarity between the Olfr29-ps1 and human OR1F2Psequence is 43%. This lncRNA promoted the immuno-suppressive function and differentiation of monocytic(Mo-)MDSCs in vitro and in vivo. It directly sponged

miR-214-3p to downregulate miR-214-3p, which may targetMyD88 to modulate the differentiation and developmentof MDSCs. The functions of Olfr29-ps1 were dependent onIL6-mediated N6-methyladenosine (m6A) modification,which not only enhanced Olfr29-ps1, but also promotedthe interaction of Olfr29-ps1 with miR-214-3p. Thus, ourresults demonstrated that the pseudogene Olfr29-ps1 mayregulate the differentiation and function of MDSCs througha m6A-modified Olfr29-ps1/miR-214-3p/MyD88 regulatorynetwork, revealing a mechanism for the regulation ofmyeloid cells and also providing potential targets for anti-tumor immunotherapy.

IntroductionMyeloid-derived suppressor cells (MDSCs) are a major regu-

lator of immune responses in cancer and inflammation. TheseMDSCs,which are derived frombonemarrowprogenitor cells, area group of heterogeneous cells with immunosuppressive func-tions, including immature granulocytes, dendritic cells, macro-phages, and early undifferentiated myeloid precursor cells.Tumors, inflammation, or infection may result in the accumula-tion and expansionofMDSCs (1–3),which are inducedbydiversecytokines such as GM-CSF, G-CSF, VEGF, and IL6 (4). These cellsare identified as CD11bþGr1þ cells, which are further dividedinto two subsets, including polymorphonuclear MDSCs(PMN-MDSC) identified as CD11bþLy6GþLy6Clo cells andmonocytic MDSCs (Mo-MDSC) identified as CD11bþLy6G–Ly6Chi

cells (5). Human MDSCs, including Mo-MDSCs (CD14þ) andPMN-MDSCs (CD15þ), are described as lineage-negative cellsthat coexpress CD11b and CD33 but lackHLA-DR.MDSCs inhibitthe immune response of T cells and mediate immunosuppression

by the expression of arginase-1 (Arg-1), NADPH oxidase 2(NOX2), nitric oxide synthase 2 (NOS2), COX2, and productionof nitric oxide (NO) and reactive oxygen species (ROS; ref. 6).

Long noncoding RNAs (lncRNA) have an important role indiverse biological processes by regulating gene expression in cis orin trans (7–9). They could be from a wide variety of transcripts,including intergenic and intragene transcripts, natural antisensechains, various enhancers, and promoter transcripts (8, 10, 11).Studies show that lncRNAs can regulate the developmentand differentiation of immune cells by a variety of mechan-isms (12–17). We have found that the lncRNA HOTAIRM1 canmodulate peripheral blood cells to differentiate into dendriticcells (DC) by sponging miR-3960 to regulate HOXA1 expres-sion (18), and have demonstrated that IL6-mediated RNCR3 andlnc-chop may affect MDSC development (19, 20). Studies havealso found that some pseudogene transcripts can function aslncRNAs to regulate related gene expression by different mechan-isms (13, 21–23). We, here, demonstrated that the pseudogeneOlfr29-ps1 may promote the immunosuppressive functionand differentiation of Mo-MDSCs by sponging miR-214-3p afterN6-methyladenosine (m6A) modification.

Materials and MethodsMice, human samples, and cell lines

C57BL/6 mice were purchased from the Beijing Animal Center(Beijing, China) and maintained in a specific pathogen-freefacility. B6.129S6-Il-6tm1Kopf (IL6–/–) and B6.SJL-CD45a(Ly5a;CD45.1) mice were purchased from the Model Animal ResearchCenter of Nanjing University (Nanjing, Jiangsu, China). OT-I orOT-II mice were from Dr. Linrong Lu (Zhejiang University). Allanimal experiments were carried out in accordance with the

1State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin,China. 2Key Laboratory of Bioactive Materials Ministry of Education, NankaiUniversity, Tianjin, China. 3Department of Immunology, Nankai University Schoolof Medicine, Nankai University, Tianjin, China.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

Corresponding Author: Rongcun Yang, Nankai University School of Medicine,Weijing Road 94#, Nankai District, Tianjin 300071, China. Phone: 86-22-23509007; Fax: 86-22-2350-9007; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-18-0443

�2019 American Association for Cancer Research.

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Nankai University Guide for the Care and Use of LaboratoryAnimals and with the approval of the Nankai University AnimalCare and Use Committee.

The peripheral blood and tissue samples from patients withcolon or rectal adenocarcinoma, which were demonstratedaccording to pathologic criteria, were obtained after informedconsent at People UnionHospital (Tianjin, China). Same age andsex healthy human peripheral blood control samples wereobtained after signing informed consents. The collection and useof all human samples (healthy individuals and patients withcolon or rectal adenocarcinoma) were approved by the Institute'sHuman Ethics Committee of Nankai University in accordancewith the Declaration of Helsinki. The samples from 12 patientswith colon cancer, 8 patients with rectal cancer, and 8 healthyindividuals (45–60 years old, male) were immediately analyzedfor peripheral blood cells and tumor tissues were stored at�70�C(not for more than 1 month). These patients met the followingcriteria: colon cancer or rectal cancer was determined by patho-logic examination and that the patients did not receive drugtreatment inside 1 month.

The murine melanoma B16, humanmonocyte cell line U937,and human embryonic kidney cell line HEK 293T cells wereobtained from the American Type Culture Collection during2013 to 2014. The murine ovarian tumor cell line 1D8 was fromDr. Richard Roden (The Johns Hopkins University School ofMedicine, gift in 2010). These cell lines were authenticated bythe short-tandem repeat method but not further authenticatedin the past years. They were not contaminated by Mycoplasmabefore or after experiments. These cells were cultured inRPMI-1640 medium with 10% FCS and 1% penicillin andstreptomycin (P/S).

Plasmids, siRNA, and microRNA transfectionA total of 1 � 107 C57BL/6 bone marrow cells (BMC) were

collected from femur and then were transfected with pcDNA3.1(5 mg/mL), pcDNA3.1-METTL3 (5 mg/mL), control scrambledsiRNA (100 nmol/L), Olfr29-ps1 siRNA (100 nmol/L), MyD88siRNA (100 nmol/L), METTL3 siRNA (100 nmol/L), miR-214-3pmimics (100 nmol/L),miR-214-3p inhibitor (100 nmol/L, chem-ically modified small RNA for cell-specific target microRNA),miR-761 mimics (100 nmol/L, chemically synthesized miRNAsequence) or scrambled control using HiPerFect transfectionreagent (Qiagen) according to the manufacturer's instructions,and then cultured in RPMI-1640 medium with 10% FCS and 1%P/S for 4 days in the presence of GM-CSF (40 ng/L) plus IL6(40 ng/L). All microRNAs, siRNAs, and control siRNAs werepurchased from Riobio. PcDNA3.1-METTL3 was generated bycloning METTL3 (ID: 56335) and conjugating into pcDNA3.1/V5-His TOPO TA vector (Invitrogen). The target sequences forOlfr29-ps1 siRNA, MyD88 siRNA and METTL3 siRNA, as wellas sources, are listed in Supplementary Table S1. For construc-ting recombinant gene-expression plasmids, the full-lengthsequence of Olfr29-ps1 (ID: 29848) or OR1F2P (ID: 26184) wasamplified using PCR methods (primer pairs are described inSupplementary Table S1). The PCR products were directly clonedinto the pcDNA3.1/V5-His TOPO TA plasmid (Invitrogen)using T4-conjugating enzyme (BioMart), which was namedM-Olfr29-ps1 or Hu-OR1F2P.

For JAK1 and STAT3 inhibitor–treated MDSCs, MDSCs, whichwere induced according to the described protocol in this method,were incubated with the JAK1 inhibitor filgotinib (20 nmol/L;

Selleckchem) or the STAT3 inhibitor HO-3867 (100 nmol/L;Selleckchem) for 24 hours.

Lentivirus construction and transductionA short hairpin RNA (shRNA) target sequence (50-GCTGTCT-

CTGTGGTTCAAA-30) of Olfr29-ps1 was chosen by BLOCK-iTRNAi Designer (Invitrogen) and/or by i-Score Designer38(https://www.med.nagoya-u.ac.jp/neurogenetics/i_Score/i_score.html). The Olfr29-ps1 shRNA constructs were made usingpGreenPuro shRNA cloning and expression lentivector kit(System Biosciences Inc.) according to the manual. The controlshNC was a luciferase control shRNA from the kit. For pack-aging lentivirus particles, the shRNA lentivector or Olfr29-ps1lentivector together with pMD2.G and psPAX2 packagingplasmids (Invitrogen) were cotransfected into 293T cells.MDSCs (1 � 107) were infected with the lentiviral supernatantsin the presence of polybrene (8 mg/mL; Millipore) by centri-fugation and then cultured with RPMI-1640 medium with 10%FCS and 1% P/S for 24 hours. The cells were then washed andcultured in the presence of GM-CSF (40 ng/mL) plus IL6(40 ng/mL) for 4 days.

In vitro induction of MDSCs and macrophagesFor in vitro induction of MDSCs, BMCs were obtained from

the femurs of C57BL/6mice and cultured in RPMI-1640mediumsupplemented with GM-CSF (40 ng/mL) only or GM-CSF(40 ng/mL) plus IL6 (40 ng/mL) for 4 days. We also inducedMDSCs in vitro as above, and thenwe harvested the cells at 0 hour,12 hours, 24 hours, and 48 hours, respectively. For the detectionof the IL6 dose-dependence of Olfr29-ps1, we set up differentconcentrations (including 10, 20, 40, and 60 ng/mL) of IL6 incombination with GM-CSF (40 ng/mL) stimulation to induceMDSC production. To prepare tumor cell supernatant-inducedCD11bþGr1þ MDSCs in vitro, 5 � 104 1D8 or B16 tumor cells(upper chamber) were cocultured with 2 � 106 BMCs (lowerchamber) in a 24-transwell plate in the presence of GM-CSF(40 ng/mL) for 4 days. CD11bþGr1þcells were sorted by FAScanor isolated using CD11b and Gr1 MACS MicroBeads and cellisolation kit (Miltenyi Biotec) according to the manufacturer'sinstructions. Macrophages were induced in vitro under the pres-ence of M-CSF (40 ng/mL) for 4 days.

Human MDSC-like cells were generated through humanperipheral blood monocytes cultured with GM-CSF and IL6according to previously reported methods (24). Human periph-eral blood mononuclear cells were isolated using a CD14þ

isolation kit (R&D Systems), and then cultured in RPMI-1640medium with 10% FCS and 1% P/S in the presence of humanrecombinant GM-CSF (40 ng/mL) and IL6 (40 ng/mL) for 4 days.

MDSCs were from C57BL/6 or C57BL/6 IL6 KO mice. B16tumor cells were injected into wild-type (WT) and IL6 KO mice.PBS was used as a control. After 4 weeks, MDSCs were sortedthrough staining CD11b and Gr1 from WT and IL6 KO mice.

M-MDSCs and PMN-MDSCs were sorted using flow cytometrythrough staining CD11b and Ly6C or Ly6G from Lv-shOlfr29-ps1–transduced MDSCs or Lv-oeOlfr29-ps1–transduced MDSCs.The tumorswere subcutaneously dissected from the groin ofmice.

Bioinformatic analysesAll miRNAs were downloaded from miRbase (miRBase V.20,

www.mirbase.org). We predicted the interactions of miRNAswith Olfr29-ps1 by two computational algorithms: RNAHybrid

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program (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid) andmiRDB (http://mirdb.org/index.html).

Flow cytometryMDSCs (1 � 106) from in vitro culture or BMCs of mice with

tumors were collected and washed twice with PBS, and thenincubated in PBS with 1% FBS blocking and antibodies for 30minutes. After washing twice with PBS, the cells were fixed in 1%paraformaldehyde and analyzed by FACScan flow cytometer(BD Biosciences). Dead cells were eliminated through 7-AAD(BD Biosciences) staining. All antibodies used in this study werelisted in Supplementary Table S1.

Real-time qPCR and PCRTotal RNA was extracted from MDSCs by using TRIzol reagent

(Life Technologies) and was transcribed to cDNA using theHiFiScript cDNA Synthesis Kit (CWBIO) according to themanufacturer's instructions. The quantitative real-time PCR(qRT-PCR) was performed by using HieffqPCR SYBR-Green Mas-ter Mix (YEASEN) in a Bio-Rad iQ5 multicolor RT-PCR system.The primers used for qRT-PCR were shown in SupplementaryTable S1. The expression of each gene, including those encodingMyD88, OR1F2P, Arg-1, iNOS, lncRNA Olfr29-ps1, and miRNAsmiR-214-3p, miR-149-5p, and miR-361-3p, was calculated usingthe 2�DDCT method. The stem-loop RT primer (BGI) was used formiR-214-3p or other miRNA reverse transcription. The relativeexpression of miRNAs was normalized to that of the internalcontrol U6. For other genes, GAPDHwas used as the endogenouscontrol. RNA content in the sample was detected using Nano-Drop. One hundred nanograms/reaction was used with threereplicates. The full-length sequence of Olfr29-ps1 was amplifiedusing an RT-PCR amplification kit (Takara; primer pairs aredescribed in Supplementary Table S1).

Western blotWestern blot was performed as described previously (25).

Briefly, cells were harvested at the indicated times and rinsedtwice with ice-cold PBS. The cells were lysed with cell-lysis buffer(Cell Signaling Technology) and centrifuged at 14,000� g for 10minutes at 4�C. The protein concentrations of the extracts weremeasured using a bicinchoninic acid assay (Pierce). Thirty micro-grams of protein was loaded into gels and then wet transferred.Hybridizationswith 1mg of primary antibodies (Abs)were carriedout for 1 hour at room temperature in blocking buffer (TBS with5% skim milk powder). Antibodies against iNOS (Cell SignalingTechnology; 1:1,000 dilution), Arg-1 (Santa Cruz Biotechnology;1:1,000 dilution), Nox2 (Santa Cruz Biotechnology; 1:1,000dilution), Cox2 (Cell Signaling Technology; 1:2,000 dilution),MyD88 (Santa Cruz Biotechnology; 1:1,000 dilution), andb-actin (Santa Cruz Biotechnology; 1:1,000 dilution) wereused. The protein–Ab complexes were detected using peroxi-dase-conjugated secondary Abs (1:5,000 dilution; BoehringerMannheim) and enhanced chemiluminescence (ECLþ; AmershamBiosciences). The signals were checked by autoradiography filmwhen the ECL substrate was added to themembranes. The primaryand secondary antibodies used in this study were listed in Supple-mentary Table S1.

Arginase activity, nitric oxide, H2O2, and ROS detectionFor arginase activity, MDSCs induced from BMCs of C57BL/6

mice (5 � 106) were lysed for 30 minutes with 100 mL of 0.1%

Triton X-100 (Sigma-Aldrich) at 4�C. Following lysis, 100 mL ofTris-HCl (25 mmol/L) and 10 mL of MnCl2 (10 mmol/L) wereadded, and the mixture was heated for 10 minutes at 56�C.Subsequently, the 100 mL lysates were incubated with 100 mLof 0.5mol/L L-arginine (Sigma-Aldrich) at 37�C for 120minutes.The reaction was stopped with 900 mL of H2SO4 (96%)/H3PO4

(85%)/H2O (1:3:7). Urea concentration was measured by absor-bance at 540 nm (Full-Wavelength Enzyme Marker, MultiskanSky) after the addition of 40 mL of 9% a-isonitrosopropiophe,followed by heating at 95�C for 30minutes. A standard curve wasgenerated using serial dilutions of 120mg/mL urea (120, 12, 1.2,and 0.12mg/mL urea). Arginase activity (unit) was defined by theamount of enzyme that catalyzes the formationof 1mgof urea perminute.

For nitric oxide production, the total nitric oxide in the 60 mLcell lysates was measured using the Nitrate/Nitrite Assay Kit(Kamiya). Equal volumes of cell lysates (60 mL), NADPH(2mmol/L, 5mL, Beyotime), FAD (10mL, Beyotime), and nitratereductase (5 mL, Beyotime) were incubated at 37�C for 30minutes, followed by the addition of 10 mL of LDH buffer(Abcom). After incubation for 30 minutes at 37�C, 50 mL ofGriess Reagent I (Beyotime) andGriess Reagent II (Beyotime) wasadded, incubated at room temperature for 10 minutes, andmeasured at 540 nm (Full-Wavelength Enzyme Marker, Multis-kan Sky).Nitrite concentrationswere quantifiedby comparing theabsorbance values with a standard curve generated by serialdilutions of 1 mol/L sodium nitrite (2, 5, 10, 20, 40, 60, and80 mmol/L).

For H2O2 production, H2O2 was evaluated using theAmplex Red Hydrogen Peroxide/Peroxidase Assay Kit(Invitrogen). Briefly, 1 � 104 MDSCs induced in vitro fromBMCs of C57BL/6 mice were resuspended in Krebs-Ringerphosphate (50 mmol/L Amplex Red reagent and 0.1 U/mLHRP). After the addition of PMA (phorbolmyristate acetate,30 ng/mL), the absorbance at 560 nm was measured using amicroplate reader at 37�C (Full-Wavelength Enzyme Marker).Absorbance values for the test samples were normalized to astandard curve generated by serial dilutions of 10 mmol/LH2O2 (0, 12.5, 25, 50, or 75 mL of 5 mmol/L H2O2 in 0.5 mLbuffer).

For ROS detection, ROS production by MDSCs was measuredby using oxidation-sensitive dye DCFDA (diacetyldichlorofluor-escein, Molecular Probes/Invitrogen) according to the reportedprotocol (26). MDSCs (1� 106) were incubated at 37�C in RPMImedium in the presence of DCFDA (2.5mmol/L) for 30minutes.For PMA-induced activation, cells were simultaneously culturedwith DCFDA and PMA (30 ng/mL), and ROS expression wasanalyzed by staining DCFDA (antibodies listed in SupplementaryTable S1).

In vitro MDSC immunosuppressive functionTo measure the immunosuppressive function of MDSCs

transduced with Olfr29-ps1 shRNA or Olfr29-ps1/lentiviruses,the splenocytes obtained from OT-I or OT-II mice were cocul-tured with MDSCs in the presence of 200 nmol/L OVA peptide(OVA257-264, GenScript) or OVA peptide (OVA323-339,GenScript) in 96-well plates at a ratio of 1:0, 1:1, 1:1/2,1:1/4, 1:1/8, and 0:1 for 48 hours. The production of IFNgwas measured by an ELISA Kit (Biotech) according to themanufacturer's instructions. For ELISA, 100 mL undilutedsupernatants were used.

Olfr29-ps1 Regulates MDSC Differentiation

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In vivo experimentsThe C57BL/6 B16 melanoma mouse model was used to

investigate the effect(s) of Olfr29-ps1–modified MDSCs ontumor growth. Mice were injected with 1 � 106 B16 cells viasubcutaneous injection at an inguinal site and were randomlydivided into Olfr29-ps1 shRNA/lentivirus, Olfr29-ps1/lentivirus(Lv-oeOlfr29-ps1), and overexpressing and knockdown controllentivirus groups (6 mice/group), and then the isolated MDSCs(1 � 106) were injected into different groups via the tail veinafter injection of tumor cells. For the preparation of Olfr29-ps1–modifiedMDSCs, 1� 107 BMCs obtained fromC57BL/6 CD45.1mice were transduced with Olfr29-ps1 shRNA or Olfr29-ps1/lentiviruses [1.5 � 108 transducing units (UT)/mL], and thenculturedwithGM-CSF (40ng/mL) plus IL6 (40ng/mL) for 4 days.The tumor volume was measured in two dimensions by calipersevery 2 days and calculated by the following formula: Width2 �Length � p/6. Twenty-four days later, the tumors were dissectedfrom the groin of the tumormice. After grinding, CD4þT cells andCD8 þ T cells, CD11bþGr1þ cells, and CD11bþLy6GþLy6Cþcellswere analyzed by flow cytometry.

RNA immunoprecipitationRNA immunoprecipitation (RIP) was performed according

to a previously reported protocol (27). Briefly, the GM-CSF plusIL6-induced MDSCs were harvested and washed. Ice-cold IP lysisbuffer (Thermo Scientific Pierce) containing 0.5% ribonucleaseinhibitor (Invitrogen) was then added, and the cells were incu-bated on ice for 5 minutes with periodic mixing. The lysates werethen transferred into a microcentrifuge tube and centrifuged at13,000 � g for 10 minutes at 4�C to pellet cell debris, and thesupernatants were transferred into a new tube, and protein Gagarose (Supplementary Table S1) was added and incubatedfor 1 hour at 4�C with rotation for preclearing. The anti–N6-methyladenosine (m6A; 1 mg, Abcam) and anti-argonaute-2antibody mouse/human (1 mg, Abcam) were added and incubat-ed overnight at 4�C with rotation. Protein G agarose was pelletedby brief centrifugation (3,000� g for 1 minute) and then washedsequentially with IP lysis buffer (containing 0.5% ribonucleaseinhibitor). Finally, RNA was extracted from protein/RNA com-plexes boundwith thebeads using TRIzol reagent anddissolved inDEPC water and quantified by quantitative PCR (qPCR) asdescribed above. The RNA IP PCR-specific primers are listed inSupplementary Table S1.

RNA–protein pulldown analysesRNA–protein pulldown analyses were performed using the

Pierce Magnetic RNA–protein pulldown Kit. MDSCs inducedin vitro from BMCs of C57BL/6 mice were harvested, and celllysates were prepared using IP lysis buffers (Thermo ScientificPierce) according to the manufacturer's protocol. Olfr29-ps1was transcribed (NEB, manual HiScribe T7 in vitro transcriptionKit) and labeled using the RNA Desthiobiotinylation Kit(Thermo Scientific Pierce) in vitro. Fifty microliters of beadsand 50 pmol/L of labeled RNA were added into RNA capturebuffer and incubated for 30 minutes at room temperature withagitation to bind labeled Olfr29-ps1 to streptavidin magneticbeads. After washing beads with an equal volume of Tris(20 mmol/L, pH 7.5), 100 mL of 1� protein–RNA bindingbuffer was added into the beads and mixed. The master mix(100 mL) of the RNA–protein binding reaction was added tothe RNA-bound beads, mixed by pipetting, and then incubated

60 minutes at 4�C with rotation to bind the proteins to RNA.After washing the beads twice with 100 mL of wash buffer,50 mL of elution buffer was added and incubated 30 minutesat 37�C with agitation. The samples obtained were used forimmunoblotting. Ago2 was used for the primary body.

Immunostaining and RNA fluorescence in situ hybridizationImmunostaining and RNA fluorescence in situ hybridization

(RNA-FISH) was performed according to the reported proto-col (20). MDSCs or human peripheral blood monocyte cellswere first slicked on sterile and 0.01% polylysine–treated slidesin the bottom of a 6-well tissue culture dish. The slides werethen processed sequentially with ice-cold CSK buffer (cytoskel-etal (CSK) buffer containing 100 mmol/L NaCl, 300 mmol/Lsucrose, 3 mmol/L MgCl2, and 10 mmol/L PIPES pH 6.8 atroom temperature, 20–25�C), 0.4% Triton X-100 buffer, andCSK buffer for 30 seconds for cell membrane perforation. Theslides were then treated with 4% PFA for 10 minutes and cold70% ethanol three times for cell fixation. After washing threetimes with ice-cold PBS, the slides were blocked in prewarmed5% goat serum (Abcom) for 30 minutes at 37�C, and the slideswere then incubated with CD11b at 37�C for 1 hour, washedthree times with 1X PBS/0.2% Tween-20 for 3 minutes on arocker, and then incubated with goat anti-Mouse IgG H&L(Abcom) at 37�C for 30 minutes. After washing three timeswith 1X PBS/0.2% Tween-20, the slides were fixed with 2% PFAat room temperature for 10 minutes. The slides were dehy-drated by moving them through a room temperature ethanolseries (85%, 95%, and 100% ethanol) for 2 minutes each, andair dried at room temperature for 15 minutes. The slides werethen hybridized using the indicated probes overnight at 37�C ina humid chamber. After washing with 2� SSC/50% formamide,2 � SSC, and 1 � SSC, each for three times, DAPI dye wasadded. Finally, the slides were sealed and then observed using aconfocal microscope (Olypus FV1000).

Dual-luciferase assayLuc-Olfr29-ps1 plasmids were constructed by cloning the

sequence of Olfr29-ps1 into the downstream of a firefly luciferasecassette in the pSiCHECK-2 vector (Promega). The primers usedwere listed in Supplementary Table S1. HEK293T cells andMDSCs were, respectively, cultured in a 24-well plate at 1 �105 cells per well. HEK293T cells (1 � 107) were cotransfectedwith Luc-Olfr29-ps1 (1 mg/mL) and 100 nmol/L miR-214-3pmimic or mimic control; MDSCs were cotransfected with luc-Olfr29-ps1 (1 mg/mL) and 100 nmol/L miR-214-3p inhibitor orinhibitor control by using Lipofectamine 2000 (Invitrogen). Aftertransfection for 24 hours, relative luciferase activity was calculatedby normalizing firefly luminescence to Renilla luminescenceusing a dual-luciferase reporter assay (Promega) according to themanufacturer's instructions (TECAN-Spark MultifunctionalEnzyme Marker).

Statistical analysesStatistical analyses were performed using two-tailed Student

t test and GraphPad Prism 5 software (GraphPad Software).Tumor growth kinetics was assessed using two-way ANOVA. TheMann–Whitney U test was used to determine significancebetween healthy individuals and patients. A 95% confidenceinterval and P < 0.05 was considered significant. �, P < 0.05;��, P < 0.01; ��, P < 0.001.

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ResultsIL6 upregulates expression of Olfr29-ps1 in MDSCs

To identify lncRNA(s) that may regulate the function anddifferentiation of MDSCs, we found that the lncRNA Olfr29-ps1was upregulated in MDSCs induced by GM-CSF and IL6 com-pared with GM-CSF alone (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc¼GSE104718; ref. 20; Fig. 1A).QRT-PCR furtherconfirmed the higher expression of Olfr29-ps1 in GM-CSF– andIL6-inducedMDSCs (Fig. 1B). This lncRNAwas distributed in thecytoplasm and nuclei of cells (Fig. 1C). Olfr29-ps1 was regulatedby proinflammatory cytokine IL6 and tumor-associated factors(Fig. 1D; Supplementary Fig. S1A and S1B). IL6-mediated expres-sion ofOlfr29-ps1was time- and dose-dependent (SupplementaryFig. S1A and S1B). Olfr29-ps1 was significantly decreased inMDSCs from B16 tumor tissues of IL6 knockout mice (Supple-mentary Fig. S1C). Tumor-associated factor–mediatedOlfr29-ps1expression was suppressed by both STAT3 and JAK3 inhibitors(Supplementary Fig. S1D), indicating that the inflammatorycytokine IL6 can upregulate Olfr29-ps1 expression.

Olfr29-ps1 is located on mouse chromosome 4 and is a pseu-dogene with a length of 963 bp (Supplementary Fig. S2A andS2B). The sequence of Olfr29-ps1 in vertebrates is conserved(Supplementary Fig. S2A), and the similarity between theOlfr29-ps1 and humanOR1F2P sequence is 43%. Human lncRNAOR1F2Pwas also detected in the humanmonocyte cell line U937(Fig. 1E). Its expression was significantly higher in human periph-eral blood mononuclear cells (HLADR–CD3–CD11bþCD33þ

cells) from colon and rectal cancer patients than in those fromhealthy individuals (Fig. 1F–H). The mouse and human lncRNAhad enrichment of histonemodificationmarkers (SupplementaryFig. S3A and S3B) and had no coding capacity (SupplementaryFig. S3C–S3E). These data suggest that Olfr29-ps1 plays a role inthe differentiation and function of MDSCs.

Olfr29-ps1 promotes differentiation of monocytic MDSCsTo investigate the effects of Olfr29-ps1 on the differentiation

and function of MDSCs, BMCs were first transduced withOlfr29-ps1 shRNA or Olfr29-ps1 lentivirus with a high transduction rate(85%–90%; Supplementary Fig. S4A–S4D) and then culturedin vitro for 4 days in the presence of GM-CSF and IL6. Olfr29-ps1knockdown decreased the percentage of CD11bþGr1þ cells sig-nificantly at 96 hours (Fig. 2A). Further studies showed thatOlfr29-ps1 knockdown reduced the percentage of monocytic(Mo)-MDSCs and increased PMN-MDSCs (Fig. 2B). These effectswere also observed in Olfr29-ps1 siRNA-transfected MDSCs(Fig. 2C–E). The overexpression of Olfr29-ps1 increased the per-centage of CD11bþGr1þ cells andMo-MDSCs, but it impeded thedifferentiation of PMN-MDSCs (Fig. 2F and G). These resultsindicated that Olfr29-ps1 is involved in the differentiation ofMDSCs and their subsets. To further confirm the effects ofOlfr29-ps1 on the differentiation of Mo-MDSCs, we next used amouseCD45.1þBMCchimeramodel. CD45.1mouseBMCs fromhomogeneous mice were transduced with Olfr29-ps1 shRNA orOlfr29-ps1/lentivirus and then injected into WT mice via the tailvein following the indicated timeline (Fig. 2H). CD45.1þ cellswere detected in the spleen at day 1 after injecting lentivirus-transduced BMCs (Fig. 2I), indicating successful establishment ofthe chimera model. In mice injected by exogenous Olfr29-ps1–transduced BMCs, the proportion of Mo-MDSCs significantlyincreased in the spleen of mice, whereas decreased Mo-MDSCs

were seen in the spleen of mice injected with Olfr29-ps1–knockdown BMCs (Fig. 2J). Taken together, our data demonstrat-ed that Olfr29-ps1 promotes the differentiation of Mo-MDSCs.

Olfr29-ps1 promotes the immunosuppressive function ofMDSCs

To analyze the effects ofOlfr29-ps1 on the immunosuppressivefunction of MDSCs, we cocultured ovalbumin (OVA)-specificOT-I or OT-II splenic cells with MDSCs. Although Olfr29-ps1–knockdownMDSCs were added into OT-I CD8þ or OT-II CD4þ Tcells, which respond toMHCI- orMHCII-restrictedOVApeptides,significantly weakened the immunosuppressive function ofMDSCs, whereasOlfr29-ps1/lentivirus–transducedMDSCs exhib-ited more suppression on IFNg production compared with con-trol MDSCs (Fig. 3A and B). The inhibition ofMDSCs on T cells isdependent on Arg-1, iNOS, NOX2, Cox2, and their products (6).Olfr29-ps1–knockdown cells had lower NO, H2O2, and ROS(Fig. 3C and D), whereas increased NO, H2O2, and ROS wereobserved in Olfr29-ps–overexpressing MDSCs compared withcontrols (Fig. 3C and D). Similar effects were also observed inOR1F2P-knockdown or -overexpressing human MDSCs (Supple-mentary Fig. S5A–S5C).Westernblot analysis showed thatOlfr29-ps1–silencing reduced protein levels of Arg-1, iNOS, Cox2,and Nox2, whereas the protein levels of Arg-1, iNOS, Cox2, andNox2 were upregulated in Olfr29-ps1–overexpressing MDSCs(Fig. 3E). Mo-MDSCs can produce high amounts of Arg-1 (28),whereas PMN-MDSCs mainly depend onH2O2 (28).Olfr29-ps1–knockdown Mo-MDSCs had decreased Arg-1, and lower H2O2

was also detected in Olfr29-ps1–knockdown PMN-MDSCs. Exog-enousOlfr29-ps1 promoted the production of Arg-1 inMo-MDSCand H2O2 in PMN-MDSCs (Fig. 3F and G). Both Olfr29-ps1–knockdown Mo-MDSCs and PMN-MDSCs also had a decreasedimmunosuppressive function, whereas Olfr29-ps1 promotedthe immunosuppressive function of both Mo-MDSCs andPMN-MDSCs (Fig. 3H and I). These results indicated thatOlfr29-ps1 promotes the immunosuppressive effect of MDSCs.

To further investigate the effects of Olfr29-ps1 on the differen-tiation and function of MDSCs in vivo, we used a murine B16melanoma model. Olfr29-ps1–knockdown or overexpressingCD45.1þ MDSCs were injected into the mice after inoculatingB16 tumor cells, and then the tumor growth was monitored.Compared with the control group, Olfr29-ps1 knockdowndecreased the immunosuppressive function of CD45.1þ MDSCs,and tumors grew slower in mice injected with Olfr29-ps1–knock-down MDSCs (Fig. 4A). A smaller tumor volume and lightertumorweightwere detected in thesemice (Fig. 4B andC),whereasOlfr29-ps1–overexpressing MDSCs caused faster tumor growth,larger tumor volume, and heavier tumor weight compared withcontrol mice (Fig. 4A–C). The mice injected with Olfr29-ps1shRNA/lentivirus–transducedMDSCs hadmoreCD4þ andCD8þ

T cells in the tumor tissues compared with the control group,whereas fewer CD4þ and CD8þ T cells appeared in the tumortissue of the mice injected with Olfr29-ps1/lentivirus–transducedMDSCs (Fig. 4D). These results indicated thatOlfr29-ps1 enhancesthe inhibition ability of MDSCs. The proportion of theCD11bþLy6G–Ly6Chi subset was reduced in the tumor of miceinjected with Olfr29-ps1–knockdown CD45.1þ MDSCs, whereasthis subset increased in the tumor withOlfr29-ps1–overexpressingCD45.1þ MDSCs (Fig. 4E), further confirming that Olfr29-ps1promoted differentiation of Mo-MDSCs. CD45.1þ cells were alsodetected in the tumor tissues and spleen in these tumor-bearing





https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE104718




Figure 1.

The expression of lncRNAOlfr29-ps1 in MDSCs.A, Heat map of the LncRNAmicroarray(GSE104718) of MDSCs induced by GM-CSF(40 ng/mL) or GM-CSF (40 ng/mL) plus IL6(40 ng/mL). The red arrow indicates the lncRNAOlfr29-ps1. B, qRT-PCR ofOlfr29-ps1 in MDSCsinduced by GM-CSFIL6, or GM-CSF plus IL6. BMC:control. C, Immunostaining and RNA-FISH inMDSCs (C2) before and (C3) after GM-CSFand IL6. C1: control probe. Scale bar, 20 mm. D,qRT-PCR ofOlfr29-ps1 in MDSCs induced byGM-CSF, GM-CSF plus IL6, GM-CSF plus B16tumor supernatant or GM-CSF plus ID8 tumorsupernatant for 4 days. E, RT-PCR ofOlfr29-ps1 inMDSCs andmacrophages, andOR1F2P in U937.Water was used a control (Ctrl). F, Flowcytometry of CD3–HLA-DR–CD33þCD11bþMDSCsin healthy individuals (n¼ 8) and patients withcolon (n¼ 12) and/or rectal cancer (n¼ 8).G,qRT-PCR ofOR1F2P in human peripheral bloodmononuclear cells from colon cancer (n¼ 12) orhealthy persons (n¼ 8). H, Immunostaining andRNA-FISH in the human colon cancer (top) and inthe peripheral blood in the patients with coloncancer (bottom). Scale bar, 100 mm. Two-tailedStudent t test was used in B and D; error bars,SEM; Mann–Whitney U test used inG; threeindependent experiments in B–Ewereperformed. �, P < 0.05; �� , P < 0.01; �� , P < 0.005.

Shang et al.





Figure 2.

Olfr29-ps1 promotes the differentiation of Mo-MDSCs.A, Flow-cytometric analysis of MDSCs transducedwith control shRNA/lentiviruses (Lv-shNC) orOlfr29-ps1 shRNA/lentiviruses (Lv-shOlfr29-ps1) at48 and 96 hours. B, Flow-cytometric analysis ofCD11bþLy6GþLy6Cþ and CD11bþLy6G–Ly6CþMDSCstransduced with control shRNA/lentiviruses(Lv-shNC) orOlfr29-ps1 shRNA/Lentiviruses(Lv-shOlfr29-ps1) at 48 and 96 hours. C, qRT-PCR ofOlfr29-ps1 in MDSCs transfected with siNC (siRNAcontrol) or siOlfr29-ps1 (Olfr29-ps1 siRNA).D and E,Flow-cytometric analysis of (D) MDSCs or (E)CD11bþLy6GþLy6Cþ and CD11bþLy6G�Ly6CþMDSCstransfected with siNC or siOlfr29-ps1. F, Flow-cytometric analysis of MDSCs transduced with emptylentivirus control (Lv-oeNC) orOlfr29-ps1 lentivirus(Lv-oeOlfr29-ps1). G, Flow-cytometric analysis ofCD11bþLy6GþLy6Cþ and CD11bþLy6G–Ly6CþMDSCsubsets after transfection with Lv-oeNC orLv-oeOlfr29-ps1. H, Schematic of in vivo experiments.After injecting genetically modified CD45.1þ BMCs(1� 107/mouse), the spleens were checked byconfocal microscopy (day 1) and flow cytometry(day 7). I, Representative images of CD45.1þ cells inthe spleen of mice indicated in H. NC, isotypicantibody. Scale bar, 50 mm. J, Flow-cytometricanalysis of Gr1þCD11bþ, CD11bþLy6GþLy6Cþ, andCD11bþLy6G–Ly6CþMDSCs in the spleens of miceafter infusing Lv-shOlfr29-ps1, Lv-oeOlfr29-ps1, andoeNC and Lv-shNC control lentivirus–treated CD45.1þ

MDSCs (6 mice/group). Two-tailed Student t test wasused; error bars, SEM; three independent experimentsin all panels were performed. NS, no significance;� , P < 0.05; �� , P < 0.01.






Figure 3.

Olfr29-ps1 promotes the suppressive function of MDSCs in vitro.A and B, The activity of T cells was measured by their capacity to produce IFNg upon OVA-MHCI–or OVA-MHCII–specific peptide stimulation (10 mg/mL) for 24 hours. IFNg in the supernatants was detected by ELISA. A, Suppressive capacity ofOlfr29-ps1shRNA/lentivirus (Lv-shOlfr29-ps1)–transduced MDSCs at the indicated ratios. B, Suppressive capacity ofOlfr29-ps1/lentivirus (Lv-oeOlfr29-ps1)–transducedMDSCs at the indicated ratios. C, NO and H2O2 production in Lv-shOlfr29-ps1–transduced MDSCs (left) and Lv-oeOlfr29-ps1–transduced MDSCs (right). D,Representative flow-cytometric histograms of ROS in Lv-shOlfr29–transduced MDSCs (left) and Lv-oeOlfr29-ps1–transduced MDSCs (right). E, Immunoblottingof Arg-1, COX2, NOX2, and iNOS inOlfr29-ps1–knockdown MDSCs and exogenousOlfr29-ps1–treated MDSCs. F, Arg-1 activity (left) and H2O2 production (right)in Lv-Olfr29-ps1–transduced Mo-MDSCs. G,Arg-1 activity (left) and H2O2 production (right) in Lv-oeOlfr29-ps1–transduced PMN-MDSCs.H and I,M-MDSCs andPMN-MDSCswere sorted from Lv-shOlfr29-ps1–transduced MDSCs or Lv-oeOlfr29-ps1–transduced MDSCs. The activity of T cells was measured as indicated inA–B. Suppressive capacity of (H) Mo-MDSCs and (I) PMN-MDSCs (T:MDSC ratio 10:1, 10 mg/mL peptides). Supernatants were analyzed after 24 hours. Two-tailedStudent t test was used. Error bars, SEM; � , P < 0.05; �� , P < 0.01; �� , P < 0.005. Three independent experiments in all panels were performed.

Shang et al.





Figure 4.

Olfr29-ps1 promotes differentiation and suppressive function of MDSCs in vivo. Tumor growth (A), tumor size (B), and tumor weight (C) in C57/BL6micebearing B16 tumors (N¼ 6/group) injected with CD45.1þMDSCs transduced withOlfr29-ps1 shRNA/lentivirus (Lv-shOlfr29-ps1) orOlfr29-ps1/lentivirus(Lv-oeOlfr29-ps1). oeNC and Lv-shNC: overexpressing and knockdown control lentiviruses, respectively.D, Flow-cytometric analysis of CD4þ or CD8þ T cells inthe tumors of mice injected with CD45.1þMDSCs transduced with Lv-shOlfr29-ps1 or Lv-oeOlfr29-ps1. E, Flow-cytometric analysis of CD45.1þGr1þCD11bþ,Gr1highCD11bþ, and Gr1lowCD11bþMDSCs in tumors of mice bearing B16 tumors after infusing Lv-shOlfr29-ps1, Lv-oeOlfr29-ps1, and oeNC and Lv-shNC controllentiviruses treated CD45.1þMDSCs. F, The representative images of CD45.1þ cells by confocal microscopy in the tumor site and spleen. NC, isotype antibody.Scale bar, 50 mm. Two-way ANOVAwas used inA; the Mann–Whitney U test was used in C; Two-tailed Student t test was used inD–F; error bars, SD; � , P < 0.05;�� , P < 0.01; �� , P < 0.005. One representative of three experiments.






mice (Fig. 4F). Taken together, these results support our findingsthat Olfr29-ps1 not only promotes the immunosuppressive func-tion but also the differentiation of Mo-MDSCs.

Olfr29-ps1–mediated effects on MDSCs depend on m6Amodification

N6-methyladenosine (m6A) is the most abundant internalmodification in eukaryotic messenger RNAs (mRNA) andlncRNAs. This modification is cell type– and condition-dependent and is reversible (29, 30). The formation of m6Aneedsmethyltransferase, includingMETTL3 (31), METTL14 (32),and WTAP (Wilms tumor 1–associated protein; ref. 33). Studieshave shown that lncRNAs can be modified by m6A (34, 35), andthe most prevalent m6A consensus sequence is GGACT (36).LncRNA Olfr29-ps1 has seven conserved sequences of GGAC thatcan potentially be modified by m6A (Fig. 5A). RIP-PCR showedthat Olfr29-ps1 was modified by m6A in MDSCs induced byGM-CSF plus IL6 (Fig. 5B). Because m6Amodification may affectthe function of mRNAs, such as mRNA splicing, transport, stabi-lization, and immune tolerance (30, 32, 37, 38), we hypothesizedthatm6Amodification could promote the formation and stabilityof Olfr29-ps1. We, thus, investigated the effects of m6A modifi-cation on Olfr29-ps1 expression. METTL3 is a methyltransferaserequired for the formation of m6A (32). Silencing METTL3reducedOlfr29-ps1 expression inMDSCs, whereas overexpressionof METTL3 increased Olfr29-ps1 in MDSCs (Fig. 5C), suggestingthat m6A modification promoted the formation and stability ofOlfr29-ps1. Further studies showed that the silencing METTL3reduced the percentage of the CD11bþLy6G–Ly6Chi subset(Fig. 5D), but overexpression of METTL3 had the oppositeeffect (Fig. 5E), suggesting that Olfr29-ps1–mediated Mo-MDSCdifferentiation is dependent on m6A modification. METTL3knockdown in MDSCs also attenuated the immunosuppressivefunction of MDSCs on OT-I and OT-II T cells, whereas the over-expression ofMETTL3 enhanced the immunosuppressive effect ofMDSCs on these T cells (Fig. 5F), indicating that Olfr29-ps1–mediated immunosuppression also depended on the m6A. Thus,these results demonstrated that Olfr29-ps1–mediated immuno-suppressive function and Mo-MDSC differentiation depends onthe m6A modification.

Olfr29-ps1–mediated effects on MDSCs is through spongingmiR-214-3p

We next investigated howOlfr29-ps1 induces immunosuppres-sive function and Mo-MDSC differentiation. Studies have shownthat lncRNAs can competitively bind to microRNAs in the cyto-plasm by acting as a competing endogenous RNA (ceRNA) and,thereby, regulate cell differentiation and function (23). BecauseOlfr29-ps1 was located in the cytoplasm, we speculated thatOlfr29-ps1–mediated immunosuppressive function and Mo-MDSC differentiation could be through ceRNAs. RNAHybridprogram and miRDB showed that Olfr29-ps1 potentially inter-acted with multiple miRNAs, such as miR-214-3p. MiR-214-3pwas significantly increased in Olfr29-psl–knockdown MDSCs(Fig. 6A). Olfr29-ps1 also had two potential binding sites formiR-214-3p (Fig. 6B). To determine whetherOlfr29-ps1–mediatedeffects on MDSCs were through sponging miR-214-3p, weperformed a dose–response experiment. The expression of miR-214-3p was gradually upregulated with increasing Olfr29-ps1shRNA/lentivirus concentration, and while Olfr29-ps1 increased,decreased miR-214-3p was also observed (Supplementary

Fig. S6A–S6C). When miR-214-3p gradually decreased with timeduring MDSC differentiation in vitro, the expression ofOlfr29-ps1was upregulated (Supplementary Fig. S6D). Transfection ofmiR-214-3p did not significantly affect the expression ofOlfr29-ps1(Supplementary Fig. S6E). Taken together, these findings demon-strated that there exists a negative correlation between Olfr29-ps1and miR-214-3p.

Next, we determined whether Olfr29-ps1 could interact withmiR-214-3p. We constructed a dual-luciferase reporter plasmidcontaining the Olfr29-ps1 sequence (luc-Olfr29-ps1), and thencotransfected luc-Olfr29-ps1 and miR-214-3p mimics into the293T cell.MiR-214-3pmimics significantly reduced the luciferaseactivity of luc-Olfr29-ps1, whereas miR-214-3p inhibitionenhanced relative luciferase activity of luc-Olfr29-ps1 (Fig. 6C),indicating that Olfr29-ps1 could interact with miR-214-3p. Next,we also determined whether m6A modification affected theinteraction of Olfr29-ps1 and miR-214-3p. Silencing METTL3significantly reduced the inhibitory function of miR-214-3p onthe luciferase activity of luc-Olfr29-ps1 (Fig. 6D). Conversely, theinhibition of miR-214-3p on the luciferase activity of luc-Olfr29-ps1was promoted by overexpressedMETTL3 (Fig. 6D), indicatingthat the m6A modification of Olfr29-ps1 is necessary for theinteraction between Olfr29-ps1 and miR-214-3p. To further vali-date the direct binding between miR-214-3p and Olfr29-ps1 atendogenous levels, we performed an anti-Ago2 RNA immuno-precipitation inMDSC extracts. As expected, bothmiR-214-3p andOlfr29-ps1 were specifically enriched in the Ago2 complexes(Fig. 6E). A RNA–protein pulldown assay further validated thespecific association betweenmiR-214-3p andOlfr29-ps1 (Fig. 6F).Ago2 enrichment was also observed in the Olfr29-ps1 pulldowncomplex (Fig. 6G), indicating that Olfr29-ps1 was recruited toAgo2-related RNA complexes and functionally interacts withmiR-214-3p. We finally tested whether Olfr29-ps1–mediated sup-pressive function and differentiation of MDSCs was throughmiR-214-3p. MiR-214-3p mimics weakened Olfr29-ps1–mediatedimmunosuppressive function and impeded the differentiationof Mo-MDSCs, whereas these functions were strengthenedby miR-214-3p inhibition (Fig. 6H–J). Taken together, our dataindicated that Olfr29-ps1 may regulate immunosuppressivefunction and Mo-MDSC differentiation through spongingmiR-214-3p.

Olfr29-ps1–mediated effects on MDSCs are through increasedMyD88

MicroRNAs regulate cell processes through regulating targetgene expression. MiR-214-3p can regulate the expression ofMyD88 (39). QRT-PCR and Western blotting showed thatmiR-214-3p reduced mRNA and protein levels of MyD88(Fig. 7A), whereas miR-214-3p inhibition increased its expres-sion (Fig. 7B), suggesting that the modulation of miR-214-3p onMDSC differentiation may be through downregulating MyD88.The expression of MyD88 in MDSCs was downregulated by theOlfr29-ps1 shRNA (Fig. 7C), but Olfr29-ps1 increased mRNAand protein levels of MyD88 (Fig. 7D). The expression patternsof Olfr29-ps1, miR-214-3p, and MyD88 in tumor MDSCs fromdifferent mice were the same as those in the MDSCs inducedin vitro (Fig. 7E–G). These results indicate that Olfr29-ps1 ispositively correlated with MyD88 expression. Previous studieshave shown that the deletion of MyD88 can regulate the differ-entiation of MDSCs (40, 41). MyD88 knockdown affected theimmunosuppressive function of MDSCs and the differentiation

Shang et al.





Figure 5.

The effect of METTL3-mediated m6Amodification on the suppressive function and differentiation of MDSCs.A,Motifs that can be modified by m6A inOlfr29-ps1.Sequences indicated in blue. B, RIP-PCR ofOlfr29-ps1 using anti-m6A in MDSCs induced by GM-CSF, IL6, or GM-CSF plus IL6. PC, positive control (Olfr29-ps1plasmids); IgG, isotype control. C, qRT-PCR ofOlfr29-ps1 in MDSCs transfected with METTL3 siRNAs (siMETTL3; left) or pcDNA-3.1-METTL3 plasmids (oeMETTL3;right). D, Representative flow cytometric (left) and group (right) analyses of CD11bþGr1þMDSCs transfected with siMETTL3 or oeMETTL3. E, Representative flowcytometric (left) and group (right) analyses of CD11bþLy6GþLy6Cþ and CD11bþLy6G–Ly6CþMDSC subsets transfected with siMETTL3 or oeMETTL3. F,Suppressive capacity of siMETTL3- or oeMETTL3-transfected MDSCs. The activity of T cells was measured by their capacity to produce IFNg upon OVA-MHCI– orOVA-MHCII–specific peptide stimulation (T:MSDC ratio 10:1, 10 mg peptides/mL). IFNg in the supernatants was detected after 24 hours by ELISA. SiNC (controlsiRNA) and oeNC (exogenous control plasmids) were used as controls. Two-tailed Student t test was used; error bars, SEM; �, P < 0.05; �� , P < 0.01; �� , P < 0.005.Three independent experiments in all panels were performed.






of Mo-MDSCs (Fig. 7H; Supplementary Fig. S7A–S7D). Thus,our data indicated that Olfr29-ps1 mediated the differentiationand functions of MDSCs are through the release of MyD88 aftersponging miR-214-3p.

We also found that two other miRNAs, not miR-214-3p, werecapable of binding to Olfr29-ps1 (Supplementary Fig. S8A–S8D).

Thismay explainwhy the effects seen bymanipulating theOlfr29-ps1 pseudogene and miRNAs were not significant. Anotherphenomenon was that although the Olfr29-ps1 pseudogene hada minor effect on Mo-MDSCs, it could affect tumor growth. Thisdichotomy was derived from tumor-mediated miR-214-3p(Supplementary Fig. S9A–S9E).

Figure 6.

Olfr29-ps1 regulates MDSC differentiationby miR-214-3p.A, qRT-PCR ofmiR-214-3p, miR-149-5p, and miR-361-3pin MDSCs transduced withOlfr29-ps1shRNA/lentivirus (Lv-shOlfr29-ps1). B,Thermodynamic energy prediction for theassociation ofOlfr29-ps1 andmiR-214-3pby RNAHybrid program. Partialsequences ofOlfr29-ps1 (top) andmiR-214-3p (bottom) are shown.Numbers above the sequences indicatethe positions of nucleotides relative to thetranscriptional start site ofOlfr29-ps1. C,Dual-luciferase reporter assay of 293Tcells cotransfected luc-Olfr29-ps1,miR-214-3p mimic (miR-214-3p), or mimiccontrol (miR-NC; left) or MDSCscotransfected luc-Olfr29-ps1 andmiR-214-3p inhibitor or inhibitor control(miR-NC; right). D, Luciferase activityof luc-Olfr29-ps1 after the addition ofmiR-214-3p mimic in MDSCs transfectedwith METTL3 siRNAs (siMETTL3; left) andpcDNA-3.1-METTL3 (oeMETTL3; right). E,Coprecipitation ofOlfr29-ps1 andmiRNAsassociated with Ago2. Anti-Ago2 RIP wasperformed in MDSC lysates. IgG, control. Fand G, Biotin-labeled RNA pulldownexperiments in MDSCs. MDSC lysateswere incubated with in vitro–synthesizedbiotin-labeled Olfr29-ps1 sense orantisense RNA, followed by qRT-PCR todetect (F) miRNAs and (G)Westernblotting to detect Ago2 associated withOlfr29-ps1. Bio-Olfr29-ps1-AS:biotinylated Olfr29-ps1-antisense RNA;bio-Olfr29-ps1-S: biotinylated Olfr29-ps1-sense RNA. H, Representative flowcytometric and (I) group analyses ofCD11bþGr1þ, CD11bþLy6GþLy6Cþ, andCD11bþLy6G–Ly6CþMDSCs. J,Suppressive capacity of MDSCs. Theactivity of T cells was measured by theircapacity to produce IFNg upon OVA-MHCI– or OVA-MHCII–specific peptidestimulation (T:MDSC ratio 10:1, 10 mgpeptides/mL). IFNg in the supernatantswas detected by ELISA after 24 hours.The MDSCs were induced by transfectingBMCs with shNC, shOlfr29-ps1 and mimicNC, shOlfr29-ps1 and miR-214-3p mimic,shOlfr29-ps1 and inhibitor NC, orshOlfr29-ps1 and miR-214-3p inhibitor,respectively. mimic NC: mimics control;inhibitor NC: inhibitor control. Two-tailedStudent t test was used; error bars, SEM;� , P < 0.05; �� , P < 0.01; �� , P < 0.005; NS,no significance. Three independentexperiments in all panels were performed.

Shang et al.





Figure 7.

Olfr29-ps1 regulates Myd88 expression by sponging miR-214-3p.A, qRT-PCR and immunoblotting analyses of MyD88 in MDSCs transfected by mimic NC ormiR-214-3p mimic. B, qRT-PCR and immunoblotting analyses of MyD88 in MDSCs transfected by inhibitor NC or miR-214-3p inhibitor. C, qRT-PCR andimmunoblotting of MyD88 in MDSCs transduced withOlfr29-ps1 shRNA/lentiviruses (Lv-shOlfr29-ps1). D, qRT-PCR and immunoblotting of MyD88 in MDSCstransfected withOlfr29-ps1/lentiviruses (Lv-oeOlfr29-ps1). E–G, qRT-PCR of (E)Olfr29-ps1, (F) miR-214-3p, and (G) MyD88 in tumor MDSCs frommice injectedwith CD45.1þMDSCs transduced with Lv-shOlfr29-ps1 orOlfr29-ps1/lentiviruses (Lv-oeOlfr29-ps1; 6 mice/group). H, Representative flow cytometric (left) andgroup (right) analyses of MDSCs transfected with MyD88 siRNA (siMyD88). SiNC: siRNA control. Two-tailed Student t test was used inA–H; error bars, SEM;� , P < 0.05; �� , P < 0.01; �� , P < 0.005. Three independent experiments in all panels were performed.






DiscussionIn this study, we identified the pseudogene lncRNAOlfr29-ps1,

which may promote the immunosuppressive function and dif-ferentiation of Mo-MDSCs. Olfr29-ps1 can sponge miR-214-3pto cause increased expression of MyD88, a target gene ofmiR-214-3p. The interaction of Olfr29-ps1 and miR-214-3p isdependent on the m6Amodification of Olfr29-ps1. We also foundthat Olfr29-ps1 was expressed in tumor MDSCs, suggesting apotential role ofOlfr29-ps1 in antitumor immunity. Thus, our datademonstrated an m6A-modified Olfr29-ps1/miR-214-3p/MyD88network to regulate the immunosuppressive function and differ-entiation of Mo-MDSCs in the inflammatory tumor environment.

Multiple data have described the presence of MDSCs inpatients with tumors, such as colon cancer, lung cancer,breast cancer, pancreatic adenocarcinomas, urothelial carci-noma, kidney cancer, and glioblastoma (28, 42). Studies haveshown that Mo-MDSCs have higher suppressive activity thanPMN-MDSCs (43). In tumor tissues, Mo-MDSCs are more prom-inent than PMN-MDSCs, and Mo-MDSCs may rapidly differen-tiate into tumor-associated macrophages (28). Mo-MDSCs canproduce high amounts of NO, Arg-1, and immune-suppressivecytokines, which have longer half-lives than the ROS producedby PMN-MDSCs (43). Less immunosuppressive PMN-MDSCsthan Mo-MDSCs have also been confirmed at the single-celllevel (28, 42). PMN-MDSCs also have a short half-life. Thus, ourdata provide insights that could help to develop novel treatmentsfor tumors through modulating the expression of Olfr29-ps1 tocontrol the differentiation of MDSCs into Mo-MDSCs.

Studies found that there exists a novel class of lncRNAs tran-scribed frompseudogeneswithmore than200nucleotides, whichare called the pseudogene lncRNAs (14, 23). Some pseudogenelncRNAs are demonstrated to control ancestral gene expression byacing as ceRNAs to sponge miRNAs, altering the stability of theancestral mRNA or affecting the promoter activity of ancestralgenes (44, 45). Ancestral genes have defects within the evolutionof the genome, such as lack of promoters, premature terminationcodons, or code-shifting mutations, resulting in pseudogenes.However, somepseudogene lncRNAs can affect other gene expres-sion. For example, Lethe, induced by TNFa, negatively regulatesNF-kB activity by binding to NF-kB–RelA to fine tune the inflam-

matory response (22). In this study, we found the pseudogenelncRNA Olfr29-ps1 could regulate the immunosuppressive func-tion and differentiation of Mo-MDSCs through competitivelybinding to miR-214-3p, thereby releasing the expression of itstarget gene MyD88 in response to inflammatory factors.

The in vitro effects of the pseudogene and miRNA expressionwere not robust and similar to those seen in vivo. LncRNAsgenerally exert their function throughmultiplemechanisms, suchas interaction with miRNAs, which can be differentially regulatedin different environments. Thus, the environmental differencesbetween the in vitro tissue culture and the in vivo tumors couldaffect the function of lncRNAs, including pseudogene Olfr29-ps1.Future experiments will need to be conducted to more fullyunderstand how lncRNAs function in different environments anddifferent experimental setups.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: R. Yang, W. ShangDevelopment of methodology: W. ShangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):W. Shang, Y. GaoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): W. ShangWriting, review, and/or revision of the manuscript: R. Yang, W. ShangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Z. Tang, Y. ZhangStudy supervision: R. Yang, Y. Zhang

AcknowledgmentsThis research was supported by the National Key Research andDevelopment

Program of China (2016YFC1303604) and NSFC grants 91842302, 91029736,9162910, 81600436, and91442111, the JointNSFC-ISF Research Program, andthe State Key Laboratory of Medicinal Chemical Biology.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 30, 2018; revised October 11, 2018; accepted March 22, 2019;published first March 26, 2019.

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2019;7:813-827. Published OnlineFirst March 26, 2019.Cancer Immunol Res Wencong Shang, Yunhuan Gao, Zhenzhen Tang, et al. and Differentiation of Monocytic MDSCs

Promotes the Suppressive FunctionOlfr29-ps1The Pseudogene

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