MIR4435-2HG, also known as LINC00978, has previously been described as an oncogenic long noncoding RNA (lncRNA). However, we show here that Mir4435-2hg depletion promoted colorectal tumorigenesis and progression in in vivo models of colitis-associated colorectal cancer, spontaneous intestinal adenomatous polyposis, and subcutaneous tumors. Alteration of MIR4435-2HG in colorectal cancer cells did not change the potential for cell proliferation, migration, or invasion in vitro. RNAscope assays showed that most MIR4435-2HG was located in the tumor stroma, which caused high expression of MIR4435-2HG in colorectal cancer tumor tissue. Transcriptome analysis of colorectal cancer tissues from wild-type and Mir4435-2hg–deficient mice revealed Mir4435-2hg as a tumor suppressor gene that regulated the immune microenvironment. Loss of Mir4435-2hg led to a decline in neutrophils and elevation of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC). In tissue-specific Mir4435-2hg knockout mice, we confirmed that Mir4435-2hg depletion in neutrophils, but not in intestinal epithelial cells, promoted colorectal cancer progression. Mechanistically, Mir4435-2hg depletion enhanced the immunosuppressive ability of PMN-MDSCs by disturbing their fatty acid metabolism. These findings suggest that MIR4435-2HG is a tumor-suppressing lncRNA whose deficiency could increase tumor-infiltrating PMN-MDSCs and enhance the immunosuppressive potential of PMN-MDSCs to promote colorectal cancer development. This provides a theoretical basis for further illustrating the pathogenesis of colorectal cancer and a potential antitumor immunotherapy target.

Long noncoding RNAs (lncRNA) are a class of over 200 nucleotide transcripts without protein-coding capacity (1). Various studies demonstrate the vital roles of lncRNAs in tumorigenesis, proliferation, metastasis, angiogenesis, and regulation of the immune microenvironment (2, 3). As the third leading cause of malignant cancer in the world (4), colorectal cancer is regulated at the initiation and progression stages by the aberrant expression and mutations of lncRNAs. Our previous studies demonstrate that LINC01133 can inhibit colorectal cancer epithelial–mesenchymal transition and metastasis by interacting with Serine And Arginine Rich Splicing Factor 6 (SRSF6; ref. 5), and high expression of the lncRNA CYTOR in tumor cells drives colorectal cancer progression (6). We also found that another novel lncRNA, MIR4435-2HG, which is highly homologous to CYTOR (7), is upregulated in colorectal cancer tumor tissue. An increasing number of publications have since then reported MIR4435-2HG as an oncogene in multiple tumor types, such as lung cancer (8, 9), hepatocellular carcinoma (10, 11), gastric cancer (12), breast cancer (13, 14), prostate cancer (15, 16), and colorectal cancer (17–19). These results were derived from analyses of clinical tissue samples or public databases and gain- or loss-of-function assays in in vitro tumor cell lines, but the roles of MIR4435-2HG in the tumor stroma or microenvironment, and its detailed regulatory mechanisms, remain unknown.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells, including pathologically activated monocytes and relatively immature neutrophils, that are generated from many pathological conditions, such as infection, inflammation, and cancer. MDSCs consist of polymorphonuclear MDSCs (PMN-MDSC) and monocyte MDSCs (M-MDSCs), whose morphology and phenotype are similar to those of neutrophils and monocytes, respectively (20, 21). PMN-MDSCs are the prevalent population of MDSCs in most types of cancer, including lung, breast, colon, and pancreatic cancer (22, 23). The salient feature of MDSCs is their inhibition of T-cell proliferation and activation by inducing arginase-1 (Arg1), reactive oxygen species production, and nitric oxide production, which culminate in the promotion of tumor progression (24, 25). MDSCs have also been reported to produce prostaglandin E2, calcium-binding proteins S100A8/S100A9, matrix metalloproteinases, IL10, TGFβ, and other cytokines to promote angiogenesis, tumor proliferation, and metastasis (26). Clinical investigation has shown that the accumulation of MDSCs positively associates with advanced cancer stage and metastatic burden in patients with colorectal cancer (27, 28).

So far, some lncRNAs, such as PVT1 (29), lnc-C/EBPβ (30), and lnc-chop (31), have been identified to regulate the immunosuppressive activity of MDSCs. However, the roles of lncRNAs in MDSCs have still not been clarified in detail. In this study, our findings repositioned MIR4435-2HG as a tumor-suppressing lncRNA in colorectal cancer. Mir4435-2hg depletion increased tumor-infiltrating PMN-MDSCs and promoted lipid accumulation in MDSCs, thus enhancing the immunosuppressive function of MDSCs and promoting tumorigenesis and progression in both colitis-associated and Apcmin/+ spontaneous colorectal cancer mouse models. These findings imply that we need to reexamine the conclusions drawn from previous MIR4435-2HG expression data and experiments in vitro.

Cell lines and culture

Colorectal cancer cell lines DLD1, HCT8, HT29, HCT116, RKO, SW480, SW620, and CT26 were purchased from the ATCC in 2011. The MC38 cell line was kindly provided by Cell Bank/Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China) in 2015. DLD1, HCT8, HCT116, HT29, SW480, SW620, CT26 cells were cultured in RPMI1640 (Gibco) and RKO, MC38 cells were cultured in DMEM (Gibco) medium according to the manufacturer's instruction, with 10% FBS (Gibco) and 1% penicillin–streptomycin (Biosharp, catalog no. BL505A) in a humidified atmosphere at 37°C with 5% CO2. All cells were frozen at passages 2 to 5 after purchase. Experiments were performed using passages 3 to 20 after removal from liquid nitrogen. Mycoplasma was test routinely using GMyc-PCR Mycoplasma Detector Kit (Yeasen). The most recent cell line authentication was in September 2018 by short tandem repeat (STR) analysis in Forensic Science Center, Zhejiang University, Hangzhou, China.

Transfection of vectors and siRNA

For overexpressing MIR4435-2HG in DLD1 and HCT116 or Mir4435-2hg in MC38 and CT26 cells, plasmids containing MIR4435-2HG (NR_015395.2) or Mir4435-2hg variant 1 (NR_028589.1), variant 2 (NR_028590.1), variant 3 (NR_028591.1), respectively, were constructed on the basis of pCDH empty backbone (WZ Biosciences Inc.) using ClonExpress II One Step Cloning Kit (Vazyme, catalog no. C112). pCDH empty vector was used as control. Plasmids were then transfected into DLD1, HCT116, MC38 or CT26 by lipo2000 (Invitrogen, catalog no. 11668019) according to the manufacturer's instructions. siRNAs targeting MIR4435-2HG (synthesized in Gemma, Supplementary Table S1) were transfected into SW480 and RKO cells using the Genmute siRNA transfection reagent (SignaGen, catalog no. SL100568) according to the manufacturer's instructions. The overexpressing or silencing effects were confirmed by RNA isolation and quantitative PCR, as detailed below.

Construction of knockout cell lines via CRISPR/Cas9

To construct MIR4435-2HG-deficient SW480 cells and Mir4435-2hg–deficient MC38 cells, 2 μg pSpCas9-BB-2A-GFP plasmid containing two sgRNAs (purchased from Genscript; sgRNA sequences are listed in Supplementary Table S1) were transfected to cells at 50% confluence in a 6-well plate by Lipofectamine 2000 according to the manufacturer's instructions. After one week, single GFP+ cells were selected by Beckman moflo Astrios EQ into wells of a 96-well plate. These single-cell clones were cultured as detailed above, and knockout was confirmed by PCR and RT-qPCR at the genome and transcriptional level. The clones without editing by Cas9 were used as mock cells. sgRNA sequences and PCR primers are listed in Supplementary Table S1.

Mice and colorectal cancer tumor models

Mir4435-2hg+/, Mir445-2hgflox/+, Villin-Cre, S100a8-Cre mice were constructed through CRISPR/Cas9 and pronuclear microinjection techniques by GemPharmatech Co., Ltd, as indicated in Supplementary Figures. Apcmin/+ mice were a gift from Dr. Rongpan Bai, Zhejiang University School of Medicine. Primer sequences for PCR genotyping are listed in Supplementary Table S1. All mice were C57BL/6J background which were bred and maintained under pathogen-free conditions at the laboratory animal center, Zhejiang University. All mice were monitored every day by the staffs of the laboratory animal center and twice one week by the authors. The protocol (ZJU20160023) for this study was approved by the Institutional Animal Care and Use Committee at Zhejiang University.

For the azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colorectal cancer model, 6-week male wild-type (WT), Mir4435-2hg−/, Mir445-2hgflox/flox, Mir445-2hgflox/flox Villin-Cre, Mir445-2hgflox/flox S100A8-Cre mice were injected intraperitoneally with 10 mg/kg AOM (Sigma-Aldrich, catalog no. A5486), and after one week, they were fed on and off 1.5% DSS (MPbio, catalog no. 160110) in the drinking water. One week of 1.5% DSS in the drinking water, followed 2 weeks of normal water constituted one DSS cycle. As indicated figures, mice were euthanatized after two DSS cycles for the short-term cohort or after three DSS cycles for the classical term cohort. For the long-term cohort, mice were fed with normal water for another 15 weeks after three DSS cycles before euthanasia. In the cohort for survival analysis, mice were fed with normal water after three DSS cycles until death. The colon was harvested, cut open along the main axis, washed with saline, and tumor numbers were counted. Tumor size was defined as the tumor diameter measured by a caliper, and tumor burden was defined as the summation of total tumor volume. Histologic examination was performed on paraffin-embedded sections after hematoxylin and eosin (H&E) staining, as detailed below.

For spontaneous intestinal adenomatous polyposis, Apcmin/+ mice were sacrificed at 4 months old. Small intestines and colons were longitudinally cut open to count tumor numbers. Histologic examination was performed on paraffin-embedded sections after H&E staining, as described below.

For subcutaneous tumor models, male WT, Mir4435-2hg−/, Mir445-2hgflox/flox, Mir445-2hgflox/flox S100A8-Cre mice aged 6–8 weeks were subcutaneously injected in the right flank with 1×105 WT MC38 cells. For Supplementary Fig. S5D, 1×106, 1×104, or 2×103 WT MC38 cell were injected as indicated in the figure. In Fig. 2H, 1×105 mock or KO MC38 cells were injected as indicted in the figure. Tumor length and width were measured with a Vernier caliper every three days. Tumor volume was calculated as length × width2/2.

Figure 1.

Mir4435-2hg deletion promotes colorectal tumorigenesis and progression. A, Meta-analysis for the expression of MIR4435-2HG in colorectal cancer tumor tissues compared to paired adjacent normal tissues for 10 datasets from TCGA and GEO. B, RNAscope assay for detecting the sublocation of MIR4435-2HG in human colorectal cancer tissues. Brown dots represent MIR4435-2HG (data representative of n = 5). Scale bar, 200 μm; zoomed, 50 μm. C, Schematic of three cohorts of AOM/DSS-induced colorectal cancer using wild-type (WT) and Mir4435-2hg−/− mice. W, week; i.p., intraperitoneal injection. D, Representative H&E staining of colorectum from WT and Mir4435-2hg−/− mice in the short-term AOM/DSS model. Scale bar, 2.5 mm; zoomed, 250 μm. Quantification of adenomas (E) and lymphoid follicles (F) in the colorectum, counted from three different H&E staining sections of one sample. Each spot represents one mouse, n = 9/group. Gross view (G) and representative H&E staining (H) of colorectum from WT and Mir4435-2hg−/− mice in the classical term AOM/DSS model, n = 6/group. Scale bar, 2.5 mm; zoomed, 250 μm. Quantification of colon tumor number (I), tumor size (J), and tumor burden (K). For I and K, each spot represents one mouse. For J, each spot represents one tumor. Gross view (L) and representative H&E staining (M) of colorectum from WT and Mir4435-2hg−/− mice in long-term AOM/DSS model, n = 5 for each group. Scale bar, 1 mm; zoomed, 250 μm. N, Quantification of colon tumor number. Each spot represents one mouse. O, Quantification of tumor size. Each spot represents one tumor. P, Percentage of mice with invasive adenocarcinomas in long-term AOM/DSS model. Q, Survival analysis of WT and Mir4435-2hg−/− mice with AOM/DSS treatment, n = 10 per group. R, Quantification of tumor number from intestine and colorectum of 4-month ApcMin/+/WT and ApcMin/+/ Mir4435-2hg−/− mice. Each spot represents one mouse, n = 13–14/group. S, Survival analysis of ApcMin/+/WT and ApcMin/+/ Mir4435-2hg −/− mice, n = 10–11/group. Data are presented as mean±SD; statistical significance was assessed by an unpaired t test (E, F, I, J, K, N, O, R) or log-rank test (Q and S; ns, P > 0.05; *, P < 0.05; **, P < 0.01).

Figure 1.

Mir4435-2hg deletion promotes colorectal tumorigenesis and progression. A, Meta-analysis for the expression of MIR4435-2HG in colorectal cancer tumor tissues compared to paired adjacent normal tissues for 10 datasets from TCGA and GEO. B, RNAscope assay for detecting the sublocation of MIR4435-2HG in human colorectal cancer tissues. Brown dots represent MIR4435-2HG (data representative of n = 5). Scale bar, 200 μm; zoomed, 50 μm. C, Schematic of three cohorts of AOM/DSS-induced colorectal cancer using wild-type (WT) and Mir4435-2hg−/− mice. W, week; i.p., intraperitoneal injection. D, Representative H&E staining of colorectum from WT and Mir4435-2hg−/− mice in the short-term AOM/DSS model. Scale bar, 2.5 mm; zoomed, 250 μm. Quantification of adenomas (E) and lymphoid follicles (F) in the colorectum, counted from three different H&E staining sections of one sample. Each spot represents one mouse, n = 9/group. Gross view (G) and representative H&E staining (H) of colorectum from WT and Mir4435-2hg−/− mice in the classical term AOM/DSS model, n = 6/group. Scale bar, 2.5 mm; zoomed, 250 μm. Quantification of colon tumor number (I), tumor size (J), and tumor burden (K). For I and K, each spot represents one mouse. For J, each spot represents one tumor. Gross view (L) and representative H&E staining (M) of colorectum from WT and Mir4435-2hg−/− mice in long-term AOM/DSS model, n = 5 for each group. Scale bar, 1 mm; zoomed, 250 μm. N, Quantification of colon tumor number. Each spot represents one mouse. O, Quantification of tumor size. Each spot represents one tumor. P, Percentage of mice with invasive adenocarcinomas in long-term AOM/DSS model. Q, Survival analysis of WT and Mir4435-2hg−/− mice with AOM/DSS treatment, n = 10 per group. R, Quantification of tumor number from intestine and colorectum of 4-month ApcMin/+/WT and ApcMin/+/ Mir4435-2hg−/− mice. Each spot represents one mouse, n = 13–14/group. S, Survival analysis of ApcMin/+/WT and ApcMin/+/ Mir4435-2hg −/− mice, n = 10–11/group. Data are presented as mean±SD; statistical significance was assessed by an unpaired t test (E, F, I, J, K, N, O, R) or log-rank test (Q and S; ns, P > 0.05; *, P < 0.05; **, P < 0.01).

Close modal

For verifying tissue-specific depletion of Mir4435-2hg, neutrophils and intestinal epithelial cells were collected from naïve Mir445-2hgflox/flox, Mir445-2hgflox/flox Villin-Cre and Mir445-2hgflox/flox S100A8-Cre mice. Neutrophils were collected from bone marrow (as described below) by flow cell sorter (Beckman moflo Astrios EQ) as labeled CD11b+Ly6G+ cells (antibodies list in Supplementary Table S2). Intestinal epithelial cells were collected by digesting colons using striping buffer containing 5 mmol/L EDTA (Invitrogen, catalog no. AM9260G), 1 mmol/L DTT (Beyotime, catalog no. ST041), 5% FBS in HBSS (Beyotime, catalog no. C0219), in 37°C 150 rpm for 40 minutes in the MQW-63R shaker. RNA isolation and quantitative PCR were then carried out as detailed below to verify the Mir4435-2hg depletion.

H&E staining

Brain, heart, kidney, liver, lung, spleen, stomach, small intestine, colon tissues from naïve 8-week male WT and Mir4435-2hg−/− mice, colon tissues from AOM/DSS-induced colorectal cancer models, and small intestine and colon tissues from 4-month Apcmin/+ mice were fixed with 10% neutral buffered formalin and paraffin embedded. After being sectioned, deparaffinized, and rehydrated, 4-μm-thick sections were stained with hematoxylin (Sigma-Aldrich, catalog no. H9627) for 10 minutes and eosin (Sigma-Aldrich, catalog no. 230251) for 2 seconds at room temperature. The slides were scanned using the NanoZoomer digital slice scanner (Hamamatsu). Tissue alterations were determined via tissue morphology, structure and cell morphology by a pathologist.

IHC

Four-micron-thick paraffin-embedded colon sections from the AOM/DSS-induced colorectal cancer models were deparaffinized in xylene (Sinopharm) and rehydrated through a graded series of ethanol solutions (Sinopharm). After blocking endogenous peroxidase activity in 3% hydrogen peroxide (ZSGB-BIO) at room temperature for 15 minutes, the tissue sections were treated with 0.01 mol/L citrate buffer (pH 6.0; OriGene) under high pressure in a pressure cooker for 3 minutes to complete antigen retrieval. Blocking was performed with 10% bovine serum (Gibco) for 30 minutes at room temperature, and then the sections were incubated with primary antibodies to CD8a (1:500) and MRP8 (1:1,000) overnight at 4°C (Supplementary Table S2), followed by incubation with 100 μL goat anti-rabbit secondary antibody (ZSGB-BIO, catalog no. PV6001) at room temperature for 30 minutes. Staining was visualized with 3,3′-diaminobenzidine (DAB) (ZSGB-BIO, catalog no. PV8000), and sections were counterstained with hematoxylin, dehydrated, and covered with a coverslip. The slides were scanned using the NanoZoomer digital slice scanner (Hamamatsu). Image analysis and quantitation were performed with ImageJ (NIH, Bethesda, MD).

Splenocytes and MC38 cell coculture assays

WT MC38 cells in logarithmic phase were trypsinized, washed twice to remove residual FBS, and then stained with 5 μmol/L CFSE (eBioscience, Cat# 65–0850) at room temperature for 10 minutes. Labeling was stopped by adding 4 to 5 volumes of cold 1640 medium containing 10% FBS, and then cells were incubated on ice for 5 minutes. After washing three times with complete medium, 2×105 CFSE-labeled MC38 cells were plated in 12-well plates with splenocytes isolated WT or Mir4435-2hg−/− mice after erythrocytes were lysed using RBC Lysis Buffer (Santa Cruz Biotechnology, catalog no. sc-296258). After incubating for 40 hours, both splenocytes and tumor cells were collected for flow cytometry analysis, as described below.

Bone marrow–derived MDSCs

Tibias and femurs from WT and Mir4435-2hg−/− mice were removed, and bone marrow (BM) cells were flushed from the bones in PBS containing 5% FBS. Erythrocytes were removed using RBC Lysis Buffer. A total of 1 × 106/mL BM cells were plated in 6-well plates in RPMI1640 complete medium containing 10% FBS, 1% penicillin–streptomycin, and 20 ng/mL GM-CSF (BioLegend, catalog no. 576304). 25% (v/v) MC38/CT26 tumor cell culture medium was added as required, which was prefiltered using 0.22-μm filters (Millipore). After 3 days, BM-derived MDSCs were collected for flow cytometry analysis, RNA isolation, and suppressive function assays, as detailed below.

Flow cytometry analysis

Cells used for flow cytometry analysis were isolated from the indicated tissues. Peripheral blood was collected from the retro-orbital plexus or submandibular vein plexus. Splenocytes were collected by dissociating the spleens with sterile forces and passing through a 40-μm cell strainer (Biosharp). BM cells were collected from tibias and femurs as described above. Erythrocytes in samples were lysed using RBC Lysis Buffer. Tumor tissues from mice were cut into small pieces and digested in RPMI1640 medium supplemented with 5% FBS, 1 mg/mL collagenase IV (Sigma-Aldrich, catalog no. C5138), 20 μg/mL hyaluronidase (Solarbio, catalog no. H8030) for 2 hours at 37°C in a shaker. Single-cell suspensions were obtained by passing through a 40-μm nylon cell strainer. Single-cell suspensions were first stained with Fc block (Biolegend, catalog no. 101320) and then treated with the indicated antibodies for 30 minutes in 4°C in the dark. All antibodies used are listed in Supplementary Table S2. 7-AAD (BD Biosciences, catalog no. 559925) was used to exclude dead cells. For intracellular staining, the Fix & Perm Kit (Multi Science, catalog no. GAS005) was used following the manufacturer's instructions. For lipid staining after surface staining, cells were resuspended in 500 μL of BODIPY 493/503 (Invitrogen, catalog no. D3922) at 1 μg/mL for 20 minutes at room temperature in the dark, and then washed three times with PBS. For reactive oxygen species (ROS) analysis, surface-stained cells were incubated with 10 μmol/L DCFH-DA probe (Beyotime, catalog no. S0033S) for 20 minutes at 37°C, and then washed three times with PBS before detection. Cells were run on Beckman CytoFLEX LX, and the data were analyzed by FlowJo 10.4 (Tristar).

RNA isolation and qPCR

Total RNA from cells or mouse colon tissues were isolated using TRIzol reagent (Invitrogen, catalog no. 15596018). Mouse colon tissues were homogenized by grinding in liquid nitrogen. For nuclear and cytoplasmic RNA fractionation, the PARIS Kit (Ambion, catalog no. AM1921) was used according to the manufacturer's instruction. cDNA was synthesized by HiScript II Reverse Transcriptase (Vazyme, catalog no. R223–01) and quantitative PCR analysis was performed by SYBR qPCR Master Mix (Vazyme, catalog no. Q711–02) according to the manufacturer's instruction in LightCycler 480 (Roche). GAPDH was detected in each experimental sample for normalization. Fold-change was calculated using the 2−ΔΔCt method. Three independent experiments were performed in triplicate. Genes and primers are listed in Supplementary Table S1.

Cell proliferation, migration, and invasion assay

Cell proliferation was measured by CCK8 reagent (Boster, catalog no.AR1160) according to the manufacturer's instructions. Briefly, 2,000 cells were seeded in 96-well plates and incubated with 100 μL culture medium. An aliquot of 10 μL CCK8 was added and incubated for 2 hours. The absorbance at 450 nm was measured to calculate the numbers of viable cells. Migration and invasion assays were performed with transwell and Matrigel chamber plates (24-well format; 8-μm pore size; Corning Costar, catalog no. 3422) as described previously (32). ImageJ software was used to measure the cell area in the bottom chamber to quantify the cells that migrated across the filter. Each measurement was performed in triplicate, and the experiments were repeated three times.

Suppressive function assay

Splenocytes were isolated from spleens of wild-type C57BL/6 mice, and erythrocytes were lysed by RBC Lysis Buffer. Splenocytes were stained with 5 μmol/L CFSE (eBioscience, catalog no. 65–0850), and then 3×105 CFSE-labeled splenocytes were cocultured with 3×104 WT or Mir4435-2hg−/− MDSCs in a round-bottom 96-well plate pre-coated with 5 μg/mL anti-CD3 (eBioscience, catalog no. 14–0031) and 5 μg/mL anti-CD28 (eBioscience, catalog no. 14–0281) at 4°C overnight. After coculturing for 72 hours, T-cell proliferation was assessed by CFSE dilution using Beckman CytoFLEX LX, and data were analyzed in FlowJo 10.4.

GEO and TCGA databases analysis

The Gene Expression Omnibus (GEO) databases were systematically and comprehensively searched (up to October 16, 2017). The search details were ‘(colorectal [All Fields] AND (adenoma [MeSH Terms] OR adenoma [All Fields]) AND 9[n_samples]:1000[n_samples]) AND “Homo sapiens”’. The inclusion criteria were as follows: (i) datasets had paired clinical sample tissues; (ii) experiment type was expression profiling. The exclusion criteria were as follows: (i) spotted cDNA (two-channel ratio data) array; (ii) patient had antitumor treatments. All available data were included in processing and analysis. The selected gene expression data were downloaded from the GEO database in SOFT format. The SOFT files were reformatted to a table separate expression matrix using a custom C script (https://github.com/wenjie1991/GEO_parser). The expression data were annotated by the gene information from NCBI (ftp://ftp.ncbi.nlm.nih.gov/gene/DATA/GENE_INFO/Mammalia/Homo_sapiens.gene_info.gz). The Cancer Genome Atlas (TCGA) colorectal cancer dataset was downloaded from UCSC Xena (https://xenabrowser.net/). ESTIMATE (Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data; ref. 33) R package was used to calculate the immune score in R software.

Colorectal cancer patient samples

Five colorectal cancer patient samples were used in the RNAscope assay, which were obtained from Sir Run Run Shaw Hospital, Zhejiang University in 2019 after informed consent by patients. The samples were formalin-fixed immediately after removal from patients and then dehydrated and paraffin-embedded. The collection and use of patient samples were approved by the Ethics Committee of department of Medicine, Zhejiang University (2016007).

RNAscope assay

The probe for MIR4435-2HG was synthesized by Advanced Cell Diagnostics (ACD lnc.). Detection of MIR4435-2HG in formalin-fixed paraffin-embedded (FFPE) human colorectal cancer tissues was performed using the RNAscope 2.0 HD Reagent Kit-Brown (ACD, catalog no. 322310) following the manufacturer's instructions. Briefly, 4-μm FFPE sections were deparaffinized, boiled with target retrieval reagent for 15 minutes, and then digested by protease at 40°C for 30 minutes, followed by hybridization for 2 hours at 40°C with Probe-MIR4435-2HG. After six steps of amplification, the probe was visualized with DAB, and cell nuclei were counterstained with hematoxylin. The slides were evaluated according to manufacturer's instructions by a pathologist.

RNA-sequencing, gene ontology functional analysis, and gene set enrichment analysis

RNA of colorectal tumor tissues from WT and Mir4435-2hg−/− long-term AOM/DSS mice, 4-month ApcMin/+/WT, ApcMin/+/ Mir4435-2hg−/− mice, and neutrophils from bone marrow of 8-week WT and Mir4435-2hg−/− mice were extracted, sequenced, and analyzed by Bioacme. The tumor tissues were stored in RNA Keeper (Vazyme, catalog no.R501) and neutrophils were stored in TRIzol in −80°C and then shipped under the protection of dry ice. Three biological replicates were used. Gene differential expression analysis was performed using the Cuffdiff program in Cufflinks package (https://github.com/cole-trapnell-lab/cufflinks) (34). The Benjamini–Hochberg false discovery rate method was applied to correct for multiple hypothesis testing. The genes with adjust P < 0.05, fold change > 1.5 or < 0.67 were defined as differentially expressed genes and were candidates for further analysis. A gene expression heatmap was generated with Heatmapper (http://www.heatmapper.ca/). Gene ontology enrichment analysis was performed using the online tool DAVID (https://david.ncifcrf.gov/). The results were visualized using the R package ggplot2 in R software. Gene set enrichment analysis (GSEA) was applied to the H (hallmark) gene sets. The gene set collection database MSigDBv7.2 (http://www.gsea-msigdb.org/gsea/downloads_archive.jsp) was used and sets with size of 15 to 500 were selected. Permutation of genes was used to generate null distribution, and all other parameters were kept as default.

Statistical analysis

A two-tailed Student t test was used to test for significant differences between two groups in GraphPad v6.0 (GraphPad Software) or SPSS v23 (SPSS Inc.). Kaplan–Meier survival analysis was performed using the software IBM SPSS Statistics v23 with the Log-rank (Mantel–Cox) test. Data in this article are presented as mean ± SD. P-value <0.05 was considered statistically significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Data availability

All data that support the findings of this study are available from the corresponding authors upon reasonable request. The raw RNA-sequencing (RNA-seq) data have been submitted to the National Center for Biotechnology Information (accession number: PRJNA777810, PRJNA779546, PRJNA779548).

MIR4435-2HG within tumor cells has no biological effect on colorectal cancer cells in vitro

To explore the function of MIR4435-2HG in colorectal cancer, we analyzed 10 public datasets from The Cancer Genome Atlas (TCGA) and eligible Gene Expression Omnibus (GEO) databases (including more than 10 paired normal–tumor colorectal cancer samples), which showed that MIR4435-2HG was upregulated in tumor tissues compared with paired adjacent normal tissues in 7 datasets but not in the other 3 datasets (Supplementary Fig. S1A). Therefore, we performed a meta-analysis to confirm that MIR4435-2HG was overexpressed in colorectal cancer (Fig. 1A). Survival meta-analysis conducted on 8 datasets revealed no significant relationship between the expression of MIR4435-2HG and overall patient survival when we set the cut-off value as the Youden index or median (P50; Supplementary Fig. S1B–S1E).

Next, we detected the expression and subcellular localization of MIR4435-2HG in colorectal cancer cell lines (Supplementary Fig. S2A). MIR4435-2HG had relatively high expression in both the nucleus and cytoplasm of SW480 and RKO cells (Supplementary Fig. S2B). We knocked-out MIR4435-2HG through CRISPR/Cas9 in SW480 cells (Supplementary Fig. S2C) and knocked-down MIR4435-2HG by siRNA in SW480 and RKO cells (Supplementary Fig. S2D). Neither knockout nor knockdown of MIR4435-2HG in colorectal cancer cells had an effect on proliferation (Supplementary Fig. S2E and S2F), migration, or invasion (Supplementary Fig. S2G and S2H). Consistent with these findings, ectopic expression of MIR4435-2HG in DLD1 and HCT116 cells (Supplementary Fig. S2I) did not change their proliferation (Supplementary Fig. S2J), migration, or invasion (Supplementary Fig. S2K). RNA scope showed high expression of MIR4435-2HG in the tumor stroma but very low expression in colorectal cancer cells and adjacent normal epithelial cells (Fig. 1B), which might clarify why MIR4435-2HG had high expression in tumor tissues but had no biological effect within colorectal cancer cells.

Mir4435-2hg deletion promotes colorectal tumorigenesis and progression

It remains unknown whether MIR4435-2HG in the tumor stroma plays biological roles in colorectal cancer development. We generated Mir4435-2hg–deficient mice by deleting the Mir4435-2hg locus through CRISPR/Cas9 (Supplementary Fig. S3A–3C). Consist with a former report (35), loss of Mir4435-2hg led to a decline of neutrophils, monocytes, and eosinophils but not lymphoid cells (Supplementary Fig. S3D–S3F). No other obvious alterations in major organ development or dyslipidemia were observed after deleting Mir4435-2hg (Supplementary Fig. S4A and S4B). We then divided the mouse colitis-associated (AOM/DSS) colorectal cancer model into three cohorts, including short-term, classical term, and long-term tumor models (Fig. 1C), which resemble human colorectal cancer progression at the molecular level, including Apc mutations and β-catenin translocation (36, 37). Although the adenoma in the colorectum from the short-term tumor model was not large enough for gross observation, H&E staining showed that Mir4435-2hg−/− mice carried more colorectal adenomas and lymphoid follicles than wild-type (WT) mice (Fig. 1DF). Through gross observation and H&E staining of the classical term model, we found more tumors, larger tumor sizes, and higher tumor burdens in Mir4435-2hg−/− mice than WT mice (Fig. 1GK). However, neither gross observation nor H&E staining of the long-term model presented a significant difference in the tumor number or tumor size between Mir4435-2hg−/− mice and WT mice (Fig. 1LO). Further histologic analysis (Fig. 1M) showed that all tumors from Mir4435-2hg−/− mice, but only 40% from WT mice, had penetrated through the muscularis mucosae into the submucosa (Fig. 1P). Another cohort of the AOM/DSS model was subjected to survival analysis (at the 30th week), and the Mir4435-2hg−/− mice exhibited worse survival (Fig. 1Q).

Next, ApcMin/+ mice, an animal model of spontaneous intestinal adenomatous polyposis (38), were crossed with Mir4435-2hg−/− mice to mimic spontaneous tumors. Higher Mir4435-2hg expression was detected in intestinal tumors than in normal tissues from ApcMin/+ mice (Supplementary Fig. S4C). Nevertheless, more intestinal tumors were observed in ApcMin/+/Mir4435-2hg−/− mice (Fig. 1R; Supplementary Fig. S4D and S4E). We also found that ApcMin/+/Mir4435-2hg−/− mice had worse prognosis than in ApcMin/+/WT mice (Fig. 1S). Taken together, these data suggest that Mir4435-2hg depletion contributes to sporadic colorectal tumorigenesis and progression and should be considered a tumor suppressor lncRNA.

MIR4435-2HG regulates colorectal cancer development by remodeling the immune microenvironment

To demonstrate how Mir4435-2hg regulated colorectal cancer progression, we performed transcriptome analysis on tumor tissues from WT and Mir4435-2hg−/− AOM/DSS mice by RNA-seq. Mir4435-2hg deletion caused 92 genes to be significantly upregulated and 138 genes to be significantly downregulated (Fig. 2A), which were further validated for their representative genes by qRT-PCR (Supplementary Fig. S5A). These differentially expressed genes were mainly enriched in immune response biological processes, including inflammatory response, neutrophil chemotaxis, immune system process, antigen processing and presentation, according to Gene Ontology (GO) enrichment analysis (Fig. 2B and C).

Figure 2.

MIR4435-2HG regulates colorectal cancer development by remodeling the immune microenvironment. A, Heat map of differentially expressed genes, determined via RNA-seq, in long-term colitis-associated (AOM/DSS) colorectal cancer tumor tissue from WT and Mir4435-2hg−/− (knockout, KO) mice, n = 3/group. B, Top downregulated biological process terms regulated by Mir4435-2hg depletion identified via gene ontology enrichment. C, Top upregulated biological process terms regulated by Mir4435-2hg depletion. D, Tumor growth curves for the subcutaneous colorectal cancer model, n = 8/group. 1 × 105 MC38 cells were subcutaneously inoculated to WT and Mir4435-2hg−/− (KO) mice. E, Tumors from WT and Mir4435-2hg−/− mice in day 22 for the model in D. F, Quantification of tumor weight for the model in D. G, Tumor growth curves for the subcutaneous colorectal cancer model, n = 8/group. Control (mock) or Mir4435-2hg−/− (KO) MC38 cells were subcutaneously inoculated to WT and Mir4435-2hg−/− mice. H, Tumors from WT and Mir4435-2hg−/− mice for the model in G. I, Quantification of tumor weight for the model in G. J, Flow cytometry analysis MC38 cell apoptosis after coculture with splenocytes from WT or Mir4435-2hg−/− mice at the indicated ratios. K, Quantification of proportion of Annexin V+ MC38 cells, n = 3/group. J and K, Data are representative of three independent experiments. Data are presented as mean ± SD; statistical significance was assessed by an unpaired t test (ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Figure 2.

MIR4435-2HG regulates colorectal cancer development by remodeling the immune microenvironment. A, Heat map of differentially expressed genes, determined via RNA-seq, in long-term colitis-associated (AOM/DSS) colorectal cancer tumor tissue from WT and Mir4435-2hg−/− (knockout, KO) mice, n = 3/group. B, Top downregulated biological process terms regulated by Mir4435-2hg depletion identified via gene ontology enrichment. C, Top upregulated biological process terms regulated by Mir4435-2hg depletion. D, Tumor growth curves for the subcutaneous colorectal cancer model, n = 8/group. 1 × 105 MC38 cells were subcutaneously inoculated to WT and Mir4435-2hg−/− (KO) mice. E, Tumors from WT and Mir4435-2hg−/− mice in day 22 for the model in D. F, Quantification of tumor weight for the model in D. G, Tumor growth curves for the subcutaneous colorectal cancer model, n = 8/group. Control (mock) or Mir4435-2hg−/− (KO) MC38 cells were subcutaneously inoculated to WT and Mir4435-2hg−/− mice. H, Tumors from WT and Mir4435-2hg−/− mice for the model in G. I, Quantification of tumor weight for the model in G. J, Flow cytometry analysis MC38 cell apoptosis after coculture with splenocytes from WT or Mir4435-2hg−/− mice at the indicated ratios. K, Quantification of proportion of Annexin V+ MC38 cells, n = 3/group. J and K, Data are representative of three independent experiments. Data are presented as mean ± SD; statistical significance was assessed by an unpaired t test (ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Close modal

To exclude the side effects caused by AOM/DSS-induced colitis, we also collected tumor tissues from ApcMin/+/Mir4435-2hg−/− and ApcMin/+/WT mice for RNA-seq analysis. Consistent with the AOM/DSS model, GO enrichment analysis of the upregulated genes showed that most genes associated with tumor immune-related biological processes, such as lymphocyte chemotaxis, cellular response to interferon-gamma, inflammatory response, and neutrophil chemotaxis (Supplementary Fig. S5C). These data further support that Mir4435-2hg might function as a tumor suppressor by regulating the immune microenvironment of colorectal cancer.

To test this hypothesis, the syngeneic murine colorectal cancer cell line MC38 was subcutaneously inoculated into Mir4435-2hg−/− and WT mice. We found that tumors grew more rapidly in Mir4435-2hg−/− mice (Fig. 2D). The volume and weight of tumors in Mir4435-2hg−/− mice were also significantly larger than those in WT mice (Fig. 2E and F), and the tumor formation rate in Mir4435-2hg−/− mice was significantly higher than that in WT mice when the inoculated cells were diluted at different concentrations (Supplementary Fig. S5D).

To further demonstrate that Mir4435-2hg in the tumor stroma, but not in tumor cells, regulated colorectal cancer development, we knocked-out Mir4435-2hg in MC38 cells with CRISPR/Cas9 (Supplementary Fig. S5E). Mir4435-2hg knockout did not change their proliferation, migration, or invasion (Supplementary Fig. S5F and S5G). Overexpression of three Mir4435-2hg variants in MC38 and CT26 cells also had little effect on proliferation and migration (Supplementary Fig. S5H–S5J). Mir4435-2hg knockout MC38 and mock knockout cells were synchronously injected into Mir4435-2hg−/− and WT mice. Knocking out Mir4435-2hg in MC38 cells did not affect tumor proliferation in either Mir4435-2hg−/− or WT mice; however, the microenvironment of Mir4435-2hg−/− mice significantly promoted tumor growth, not only of mock but also of Mir4435-2hg knockout MC38 cells (Fig. 2G). A similar phenotype was also observed for tumor volume and weight (Fig. 2H and I). Next, we separated splenocytes from Mir4435-2hg−/− and WT mice to coculture with MC38 cells. As expected, the apoptosis of MC38 cells was greater when they were cocultured with splenocytes, but the MC38 cell apoptosis rate was decreased by the splenocytes from Mir4435-2hg−/− mice compared with WT mice (Fig. 2J and K).

We also reanalyzed survival data from TCGA and GEO databases. The ESTIMATE algorithm was used to calculate the immune score for each sample, and samples with an immune score higher than 1,000 were selected for survival analysis. In these high immune infiltrate samples, higher expression of MIR4435-2HG tended to associate with a better prognosis, although not statistically significant (Supplementary Fig. S6). Overall, these data indicated that MIR4435-2HG might play an anticancer role by remodeling the colorectal cancer immune microenvironment.

Mir4435-2hg depletion increases PMN-MDSCs in colorectal cancer

Mir4435-2hg can control the lifespan of eosinophils, neutrophils, and monocytes through allele-specific suppression of Bim expression (35). However, it remains unknown which type of cell in the colorectal cancer stroma expresses Mir4435-2hg. Therefore, we performed an RNAscope assay in human colorectal cancer tissues, which showed that MIR4435-2HG was mainly located in neutrophils (Fig. 3A). Next, we monitored the number variations of main leukocytes in peripheral blood during the growth of subcutaneous tumors. Consistent with previous reports (39), neutrophils, monocytes, and eosinophils were increased and lymphocytes were decreased in tumor-bearing mice. Monocytes and eosinophils in Mir4435-2hg−/− mice remained lower than those in WT mice, but the number of neutrophils in Mir4435-2hg−/− mice gradually reached the same frequency as that in WT mice as tumors grew (Fig. 3B). A similar phenotype was observed in splenocytes (Supplementary Fig. S7A).

Figure 3.

Mir4435-2hg depletion increases PMN-MDSCs. A, RNAscope assay showing MIR4435-2HG localization in tumor-infiltrating neutrophils in human colorectal cancer tissues. Representative image of five samples. Neutrophils were identified by their polymorphonuclear morphology. Brown signals represent MIR4435-2HG. Scale bar, 50 μm; zoomed, 10 μm. B, Variations in proportions of neutrophils, monocytes, eosinophils, and lymphocytes in peripheral blood of WT and Mir4435-2hg−/− (KO) mice at the indicated timepoints for subcutaneous MC38 colorectal cancer tumors, n = 3/group. C, Flow cytometry splenic neutrophil apoptosis from tumor-free WT and Mir4435-2hg−/− (KO) mice, and subcutaneous MC38 tumor-bearing wild-type (WT_TB) and Mir4435-2hg−/− (KO_TB) mice, pregated on CD11b+Ly6G+ cells. D, Quantification of Annexin V+ neutrophils for mice in C, n = 3/group. E, Frequency of Annexin V+ neutrophils in in vitro cultured bone marrow (BM) cells from WT or KO mice with/without MC38 cultured medium (CM), n = 3/group. F, BIM protein expression assessed by flow cytometry in splenic neutrophils from tumor-free WT and Mir4435-2hg−/− (KO) mice, and subcutaneous MC38 tumor-bearing wild-type (WT_TB) and Mir4435-2hg−/− (KO_TB) mice. G, Quantification of BIM mean fluorescence intensity (MFI) from mice in (F), n = 3/group. H, Relative Bcl2l1 mRNA expression in BM-derived MDSCs from WT or KO mice with/without MC38 CM, n = 3/group. I, BCL2L1 protein expression assessed by flow cytometry in splenic neutrophils from tumor-free WT and Mir4435-2hg−/− (KO) mice, and subcutaneous MC38 tumor-bearing wild-type (WT_TB) and Mir4435-2hg−/− (KO_TB) mice. J, Quantification of BCL2L1 MFI for mice in (I), n = 4/group. K, Representative flow cytometry of CD14+ neutrophils from spleen of tumor-free WT and Mir4435-2hg−/− (KO) mice, and subcutaneous MC38 tumor-bearing wild-type (WT_TB), Mir4435-2hg−/− (KO_TB) mice, pregated on CD11b+Ly6G+ cells. L, Quantification of CD14+ neutrophils from K, n = 4 per group. BL, Data are representative of three independent experiments. Data are presented as mean ± SD; statistical significance was assessed by an unpaired t test (ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Figure 3.

Mir4435-2hg depletion increases PMN-MDSCs. A, RNAscope assay showing MIR4435-2HG localization in tumor-infiltrating neutrophils in human colorectal cancer tissues. Representative image of five samples. Neutrophils were identified by their polymorphonuclear morphology. Brown signals represent MIR4435-2HG. Scale bar, 50 μm; zoomed, 10 μm. B, Variations in proportions of neutrophils, monocytes, eosinophils, and lymphocytes in peripheral blood of WT and Mir4435-2hg−/− (KO) mice at the indicated timepoints for subcutaneous MC38 colorectal cancer tumors, n = 3/group. C, Flow cytometry splenic neutrophil apoptosis from tumor-free WT and Mir4435-2hg−/− (KO) mice, and subcutaneous MC38 tumor-bearing wild-type (WT_TB) and Mir4435-2hg−/− (KO_TB) mice, pregated on CD11b+Ly6G+ cells. D, Quantification of Annexin V+ neutrophils for mice in C, n = 3/group. E, Frequency of Annexin V+ neutrophils in in vitro cultured bone marrow (BM) cells from WT or KO mice with/without MC38 cultured medium (CM), n = 3/group. F, BIM protein expression assessed by flow cytometry in splenic neutrophils from tumor-free WT and Mir4435-2hg−/− (KO) mice, and subcutaneous MC38 tumor-bearing wild-type (WT_TB) and Mir4435-2hg−/− (KO_TB) mice. G, Quantification of BIM mean fluorescence intensity (MFI) from mice in (F), n = 3/group. H, Relative Bcl2l1 mRNA expression in BM-derived MDSCs from WT or KO mice with/without MC38 CM, n = 3/group. I, BCL2L1 protein expression assessed by flow cytometry in splenic neutrophils from tumor-free WT and Mir4435-2hg−/− (KO) mice, and subcutaneous MC38 tumor-bearing wild-type (WT_TB) and Mir4435-2hg−/− (KO_TB) mice. J, Quantification of BCL2L1 MFI for mice in (I), n = 4/group. K, Representative flow cytometry of CD14+ neutrophils from spleen of tumor-free WT and Mir4435-2hg−/− (KO) mice, and subcutaneous MC38 tumor-bearing wild-type (WT_TB), Mir4435-2hg−/− (KO_TB) mice, pregated on CD11b+Ly6G+ cells. L, Quantification of CD14+ neutrophils from K, n = 4 per group. BL, Data are representative of three independent experiments. Data are presented as mean ± SD; statistical significance was assessed by an unpaired t test (ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Close modal

Neutrophils in tumor-bearing mice are heterogenetic, including both classical neutrophils and PMN-MDSCs (40, 41). From our data above, we assumed that Mir4435-2hg could not control the lifespan of PMN-MDSCs and that the elevated neutrophils in tumor-bearing Mir4435-2hg−/− mice were mainly PMN-MDSCs. We, thus, detected the apoptosis of neutrophils in tumor-free and tumor-bearing WT and Mir4435-2hg−/− mice. Tumor-free Mir4435-2hg−/− mice had significantly more neutrophil apoptosis than the WT mice. However, in tumor-bearing mice, neutrophil apoptosis was reduced in both Mir4435-2hg−/− and WT mice, which may result from the GM-CSF produced by MC38 cells (42), and in tumor-bearing Mir4435-2hg−/− mice, neutrophil apoptosis rate was reduced to the same level as that in tumor-bearing WT mice (Fig. 3C and D). In BM-derived PMN-MDSCs, we also observed a decreased apoptosis rate after treatment with MC38 tumor cell culture medium, especially in Mir4435-2hg−/− cells (Fig. 3E).

Because Mir4435-2hg regulates neutrophil apoptosis by suppressing Bim expression, we detected the expression of BIM protein in neutrophils. BIM was upregulated in both tumor-free and tumor-bearing Mir4435-2hg−/− mice, and bearing tumors did not alter the expression of BIM (Fig. 3F and G; Supplementary Fig. S7B). The Bcl2 family regulates apoptosis by forming homodimers or heterodimers. Therefore, we detected alterations in all anti-apoptotic Bcl2 family members. Among them, only Bcl2l1 in BM-derived PMN-MDSCs from either WT or Mir4435-2hg−/− mice was upregulated by stimulation with MC38 tumor cell culture medium (Fig. 3H), whereas Bcl2 and Bcl2a1a were downregulated, and Bcl2l2 and Mcl1 were unaltered (Supplementary Fig. S7C). Flow cytometry also showed elevated expression of BCL2L1 in neutrophils from both WT and Mir4435-2hg−/− tumor-bearing mice (Fig. 3I and J). These results indicated that the elevated expression of BCL2L1 in PMN-MDSCs might antagonize the function of the increased BIM in Mir4435-2hg−/− mice, helping the neutrophils in tumor-bearing Mir4435-2hg−/− mice recover to the same level as in WT mice and leading to a higher proportion of PMN-MDSCs.

Single-cell RNA-seq analysis (43) has identified CD14 as a marker of PMN-MDSCs for distinguishing classical neutrophils in tumor-bearing mice. In our study, a significantly higher proportion of CD14+ cells was observed in splenic neutrophils from tumor-bearing Mir4435-2hg−/− mice than in those from WT mice. Even in tumor-free mice, Mir4435-2hg−/− mice also carried a higher proportion of CD14+ neutrophils than WT mice (Fig. 3K and L). We also investigated several types of tumor-infiltrating immune cells within subcutaneous tumors by flow cytometry. A higher proportion of neutrophils was detected in Mir4435-2hg−/− mice than in WT mice (Fig. 4A). Within the neutrophil compartment, the percentage of CD14+ cells reached average 85.7% in WT mice and 93.0% in Mir4435-2hg−/− mice (Fig. 4B), which demonstrated that tumor-infiltrating neutrophils were mostly PMN-MDSCs that were increased within the tumors of Mir4435-2hg−/− mice. In contrast, CD3+ T cells and CD8+ T cells were decreased (Fig. 4C and D). Monocytes/M-MDSCs were also significantly decreased in Mir4435-2hg−/− mice, whereas total CD45+ cells, macrophages, and M2 macrophages were unchanged (Fig. 4EH). Similar results were observed in the AOM/DSS colorectal cancer model; tumor-infiltrating PMN-MDSCs were significantly increased, and CD8+ T cells were decreased (Fig. 4I) in Mir4435-2hg−/− mice compared with WT mice by IHC. In the splenocytes/MC38 coculture system, we also observed that PMN-MDSCs were increased in the Mir4435-2hg−/− group (Supplementary Fig. S7D). These data demonstrate that loss of Mir4435-2hg could cause an increase in PMN-MDSCs, which contribute to an immunosuppressive environment.

Figure 4.

Mir4435-2hg depletion leads to increased tumor-infiltrating PMN-MDSCs and decreased T cells. Flow cytometry of tumor-infiltrating total neutrophils (A), CD14+ PMN-MDSCs (B), CD3+ T cells (C), CD8+ T cells (D), monocytes/M-MDSCs (E), total CD45+ cells (F), macrophages (G), and M2 macrophages (H) in subcutaneous MC38 tumors, n = 3–8/group. Data are representative of three independent experiments. I, Left, representative IHC of tumor-infiltrating PMN-MDSCs (S100a8) and CD8+ T cells (CD8) in the long-term AOM/DSS model. Scale bar, 100 μm. Right, quantification, n = 5/group. Data are presented as mean ± SD; statistical significance was assessed by an unpaired t test (ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 4.

Mir4435-2hg depletion leads to increased tumor-infiltrating PMN-MDSCs and decreased T cells. Flow cytometry of tumor-infiltrating total neutrophils (A), CD14+ PMN-MDSCs (B), CD3+ T cells (C), CD8+ T cells (D), monocytes/M-MDSCs (E), total CD45+ cells (F), macrophages (G), and M2 macrophages (H) in subcutaneous MC38 tumors, n = 3–8/group. Data are representative of three independent experiments. I, Left, representative IHC of tumor-infiltrating PMN-MDSCs (S100a8) and CD8+ T cells (CD8) in the long-term AOM/DSS model. Scale bar, 100 μm. Right, quantification, n = 5/group. Data are presented as mean ± SD; statistical significance was assessed by an unpaired t test (ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

Close modal

Loss of Mir4435-2hg enhances the immunosuppressive potential of PMN-MDSCs

To investigate the biological mechanism by which Mir4435-2hg regulated PMN-MDSCs, we performed RNA-seq of neutrophils from BM, which showed 429 downregulated genes and 336 upregulated genes (Fig. 5A). GO enrichment analysis showed that the downregulated genes were enriched in mitosis-associated biological processes, including cell division, mitotic nuclear division, and regulation of mitotic centrosome separation (Supplementary Fig. S8A). Because MDSCs are considered relatively immature myeloid cells, these results might indicate a disordered process of neutrophil maturation in Mir4435-2hg−/− mice, which provides another perspective from which to understand the decline in neutrophils and the elevation of PMN-MDSCs in Mir4435-2hg−/− mice.

Figure 5.

Loss of Mir4435-2hg enhances the immunosuppressive potential of PMN-MDSCs. A, Heat map of differentially expressed genes, determined via RNA-seq, in BM neutrophils from naïve 8-week WT and Mir4435-2hg−/− (KO) mice, n = 3/group. B, Top upregulated biological process terms regulated by Mir4435-2hg depletion in BM neutrophils. GSEA for cholesterol homeostasis (C) and (D) fatty acid metabolism. Flow cytometry (E) and quantification of lipid accumulation (BODIPY staining; F) of splenic neutrophil from tumor-free WT and Mir4435-2hg−/− (KO) mice, and PMN-MDSCs from subcutaneous MC38 tumor-bearing wildtype (WT_TB) and Mir4435-2hg−/− (KO_TB) mice 20 days after MC38 cells injected, n = 3/group, pregated on CD11b+Ly6G+ cells. MFI, mean fluorescence intensity. G, Lipid accumulation (BODIPY staining) of WT and Mir4435-2hg−/− BM-derived PMN-MDSCs with or without the stimulation of CT26 or MC38 cultured medium, n = 3/group. Relative Arg1 (H), Nos2 (I), and Cox2 mRNA expression (J) in BM-derived MDSCs from cultures in G, assessed by qRT-PCR, n = 3/group. K, Flow cytometry of ROS (DCFH-DA probe) in BM-derived PMN-MDSCs from cultures in G, pregated on CD11b+Ly6G+ cells. L, CFSE flow cytometry analysis of T-cell and CD8+ T-cell proliferation after coculture with WT or Mir4435-2hg−/− MDSCs for 72 hours, n = 3/group. EL, Data are representative of three independent experiments. Data are presented as mean ± SD; statistical significance was assessed by an unpaired t-test. ns, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5.

Loss of Mir4435-2hg enhances the immunosuppressive potential of PMN-MDSCs. A, Heat map of differentially expressed genes, determined via RNA-seq, in BM neutrophils from naïve 8-week WT and Mir4435-2hg−/− (KO) mice, n = 3/group. B, Top upregulated biological process terms regulated by Mir4435-2hg depletion in BM neutrophils. GSEA for cholesterol homeostasis (C) and (D) fatty acid metabolism. Flow cytometry (E) and quantification of lipid accumulation (BODIPY staining; F) of splenic neutrophil from tumor-free WT and Mir4435-2hg−/− (KO) mice, and PMN-MDSCs from subcutaneous MC38 tumor-bearing wildtype (WT_TB) and Mir4435-2hg−/− (KO_TB) mice 20 days after MC38 cells injected, n = 3/group, pregated on CD11b+Ly6G+ cells. MFI, mean fluorescence intensity. G, Lipid accumulation (BODIPY staining) of WT and Mir4435-2hg−/− BM-derived PMN-MDSCs with or without the stimulation of CT26 or MC38 cultured medium, n = 3/group. Relative Arg1 (H), Nos2 (I), and Cox2 mRNA expression (J) in BM-derived MDSCs from cultures in G, assessed by qRT-PCR, n = 3/group. K, Flow cytometry of ROS (DCFH-DA probe) in BM-derived PMN-MDSCs from cultures in G, pregated on CD11b+Ly6G+ cells. L, CFSE flow cytometry analysis of T-cell and CD8+ T-cell proliferation after coculture with WT or Mir4435-2hg−/− MDSCs for 72 hours, n = 3/group. EL, Data are representative of three independent experiments. Data are presented as mean ± SD; statistical significance was assessed by an unpaired t-test. ns, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Close modal

Most upregulated genes associated with the cholesterol biosynthetic process (Fig. 5B), and GSEA also showed that these genes were enriched in cholesterol homeostasis and fatty acid metabolism (Fig. 5C and D). Because accumulation of lipids is a critical regulator of the immunosuppressive function of PMN-MDSCs (44, 45), we presumed that higher lipid accumulation caused a stronger immunosuppressive effect of PMN-MDSCs in Mir4435-2hg−/− mice. Through BODIPY lipid staining, we observed an increasing trend of lipid accumulation in neutrophils from tumor-free Mir4435-2hg−/− mice, although the difference was not significant. Nevertheless, the PMN-MDSCs from the spleens of tumor-bearing Mir4435-2hg−/− mice had more lipids than those of WT mice (Fig. 5E and F). Consistent with this, Mir4435-2hg−/− mouse BM-derived PMN-MDSCs induced by CT26 and MC38 in vitro had more lipids than those from WT mice (Fig. 5G). Mir4435-2hg−/− mouse-derived PMN-MDSCs expressed higher Arg1, Nos2, Cox2, and ROS (Fig. 5HK), which resulted in a stronger inhibitory effect on the proliferation of T cells (Fig. 5L). Altogether, these data illustrate that loss of Mir4435-2hg enhances the immunosuppressive ability of PMN-MDSCs.

Neutrophil-specific deletion of Mir4435-2hg promotes colorectal cancer progression

To further confirm that Mir4435-2hg depletion in neutrophils, but not in intestinal epithelial cells, caused the phenotypes observed in the mouse models, we generated conditional knockout Mir4435-2hgflox/flox mice and crossed them with S100a8-Cre mice or Villin-Cre mice to construct neutrophil- or intestine-specific Mir4435-2hg deletion mice (Fig. 6A; Supplementary Fig. S8B and S8C). Flow cytometry analysis of leukocytes in peripheral blood showed a decrease in neutrophils in Mir4435-2hgflox/flox S100a8-Cre mice compared to Mir4435-2hgflox/flox and Mir4435-2hgflox/flox Villin-Cre mice, and the number of eosinophils was also significantly decreased in Mir4435-2hgflox/flox S100a8-Cre mice (Supplementary Fig. S8D). The subcutaneous tumor model showed that tumors grew significantly faster in Mir4435-2hgflox/flox S100a8-Cre mice than in control mice (Fig. 6B), and tumor volume and weight (Fig. 6C and D) were significantly increased in neutrophil-deficient Mir4435-2hg mice. Consistent with the Mir4435-2hg−/− mice, we found a higher lipid accumulation in the PMN-MDSCs from tumor-bearing Mir4435-2hgflox/flox S100a8-Cre mice (Fig. 6E), as well as in BM-derived PMN-MDSCs induced by MC38 cultured medium in vitro (Fig. 6F).

Figure 6.

Neutrophil-specific deletion of Mir4435-2hg promotes colorectal cancer progression. A, Relative Mir4435-2hg expression in neutrophils and intestinal epithelial cells from naïve 8-week Mir4435-2hgflox/flox, Mir4435-2hgflox/flox Villin-Cre, and Mir4435-2hgflox/flox S100a8-Cre mice, n = 2–3/group. B, Tumor growth curves for the subcutaneous MC38 colorectal cancer model, n = 8/group, tumors were harvested at day 22. C, Tumors from Mir4435-2hgflox/flox and Mir4435-2hgflox/flox S100a8-Cre mice from B. D, Quantification of tumor weight from mice in B. E, Flow cytometry of lipid accumulation of splenic neutrophil from tumor-free or subcutaneous MC38 tumor-bearing Mir4435-2hgflox/flox and Mir4435-2hgflox/flox S100a8-Cre mice, n = 5/group. F, Lipid accumulation in the bone marrow-derived PMN-MDSCs from the indicated mice with/without MC38 culture medium (CM), n = 3/group. G, Gross view of colorectum from Mir4435-2hgflox/flox, Mir4435-2hgflox/flox Villin-Cre, and Mir4435-2hgflox/flox S100a8-Cre mice in AOM/DSS model, n = 6/group. H, Quantification of tumor number from mice in (G); each spot represents one mouse. I, Quantification of tumor size from mice in G; each spot represents one tumor. J, Quantification of tumor burden from mice in G; each spot represents one mouse. K, Left, representative IHC of tumor-infiltrating PMN-MDSCs (S100a8) and CD8+ T cells (CD8). Scale bar, 100 μm. Right, quantification, n = 5/group. (A, E, and F) Data are representative of three independent experiments. Data are presented as mean ± SD; statistical significance was assessed by an unpaired t test (ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Figure 6.

Neutrophil-specific deletion of Mir4435-2hg promotes colorectal cancer progression. A, Relative Mir4435-2hg expression in neutrophils and intestinal epithelial cells from naïve 8-week Mir4435-2hgflox/flox, Mir4435-2hgflox/flox Villin-Cre, and Mir4435-2hgflox/flox S100a8-Cre mice, n = 2–3/group. B, Tumor growth curves for the subcutaneous MC38 colorectal cancer model, n = 8/group, tumors were harvested at day 22. C, Tumors from Mir4435-2hgflox/flox and Mir4435-2hgflox/flox S100a8-Cre mice from B. D, Quantification of tumor weight from mice in B. E, Flow cytometry of lipid accumulation of splenic neutrophil from tumor-free or subcutaneous MC38 tumor-bearing Mir4435-2hgflox/flox and Mir4435-2hgflox/flox S100a8-Cre mice, n = 5/group. F, Lipid accumulation in the bone marrow-derived PMN-MDSCs from the indicated mice with/without MC38 culture medium (CM), n = 3/group. G, Gross view of colorectum from Mir4435-2hgflox/flox, Mir4435-2hgflox/flox Villin-Cre, and Mir4435-2hgflox/flox S100a8-Cre mice in AOM/DSS model, n = 6/group. H, Quantification of tumor number from mice in (G); each spot represents one mouse. I, Quantification of tumor size from mice in G; each spot represents one tumor. J, Quantification of tumor burden from mice in G; each spot represents one mouse. K, Left, representative IHC of tumor-infiltrating PMN-MDSCs (S100a8) and CD8+ T cells (CD8). Scale bar, 100 μm. Right, quantification, n = 5/group. (A, E, and F) Data are representative of three independent experiments. Data are presented as mean ± SD; statistical significance was assessed by an unpaired t test (ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Close modal

In the AOM/DSS model, Mir4435-2hgflox/flox S100a8-Cre mice developed more and larger tumors than Mir4435-2hgflox/flox Villin-Cre and Mir4435-2hgflox/flox mice (Fig. 6G; Supplementary Fig. S8E). Although the tumor counts were not significantly higher in Mir4435-2hgflox/flox S100a8-Cre mice (Fig. 6H), the average tumor size and tumor burden (Fig. 6I and J) were significantly increased in Mir4435-2hgflox/flox S100a8-Cre mice. No obvious changes in tumor count, tumor size, or tumor burden were observed between Mir4435-2hgflox/flox Villin-Cre mice and Mir4435-2hgflox/flox mice. We also observed more tumor-infiltrating PMN-MDSCs and fewer CD8+ T cells in Mir4435-2hgflox/flox S100a8-Cre mice than in Mir4435-2hgflox/flox Villin-Cre mice and Mir4435-2hgflox/flox mice (Fig. 6K). Overall, deletion of Mir4435-2hg in neutrophils/PMN-MDSCs, but not in the intestinal epithelium, promoted colorectal cancer progression.

Here, genetically engineered mouse models demonstrated that MIR4435-2HG suppressed colorectal cancer initiation and progression by regulating the immunosuppressive activity of PMN-MDSCs. These findings are a significant departure from the literature, especially the colorectal cancer literature. Ouyang and colleagues (17) report MIR4435-2HG as a poor prognostic marker in their analysis of TCGA and two selected GEO databases. However, their analysis did not consider the high homology of MIR4435-2HG with CYTOR, which could have resulted in a false expression value for MIR4435-2HG that included CYTOR expression (TCGA anlaysis). In the GEO database, probe 232918_at is designed as a specific probe targeting MIR4435-2HG, whereas probe 225799_at targets both MIR4435-2HG and CYTOR. The previous study (17) used probe 225799_at to evaluate the expression value of MIR4435-2HG. In this study, we used the signal value of 232918_at to determine expression of MIR4435-2HG. We also downloaded all eligible GEO databases as of October 2017 to perform a meta-analysis, and the results revealed no significant relationship between MIR4435-2HG expression and overall survival. Shen and colleagues (18) investigated MIR4435-2HG expression in clinical colorectal cancer samples, but the primers utilized to detect MIR4435-2HG do not match MIR4435-2HG sequence. Another study reports that MIR4435-2HG promotes colorectal cancer proliferation and metastasis through the miR-206/YAP1 axis (19), but both the primers and the shRNA targeting MIR4435-2HG used also target CYTOR. Therefore, we believe the conclusions from these studies may not reflect the true expression of MIR4435-2HG and that the phenotypes observed may have been confounded by alterations in CYTOR expression. Other studies of MIR4435-2HG in other types of cancer may also need to be reexamined to verify whether off-target effects occurred. In the meanwhile, MIR4435-2HG might have different distribution and diverse functions in different tumor types.

In our study, we used specific CRISPR-Cas9/sgRNAs, siRNAs, and an overexpression vector to alter the expression of MIR4435-2HG in colorectal cancer cells, which did not affect CYTOR expression (Supplementary Fig. S9). Alteration of MIR4435-2HG did not change the proliferation, migration, or invasion of human colorectal cancer cells. The same phenotypes were also observed in colorectal cancer cell lines from mice. In mice, CYTOR is deficient (no homologous sequence found). Because of the high level of MIR4435-2HG in the tumor stroma and the loss of CYTOR in mice, we generated Mir4435-2hg–deficient mice. Mir4435-2hg-specific depletion promoted colorectal tumorigenesis and progression in a colitis-associated colorectal cancer model, spontaneous intestinal adenomatous model, and subcutaneous tumor experiments. Although no significant change in the tumor number or tumor size was observed in the long-term colitis-associated colorectal cancer model using Mir4435-2hg−/− mice, H&E staining showed that tumors in Mir4435-2hg−/− mice exhibited higher invasion potency. The reason might be that tumor formation and proliferation reached a maximum for long-term induction, but Mir4435-2hg depletion still promoted colorectal cancer progression in later stages. In Mir4435-2hg−/− mice, BIM upregulation caused neutrophil apoptosis, but PMN-MDSCs with high BCL2L1 expression resisted BIM-induced apoptosis. The increased lipid accumulation in Mir4435-2hg−/− PMN-MDSCs enhanced their immunosuppressive activities. Therefore, we observed higher infiltration of PMN-MDSCs and fewer T cells in tumors from Mir4435-2hg−/− mice. In splenocyte/MC38 co-culture assays, we also observed increased PMN-MDSCs in the Mir4435-2hg−/− group, but possibly due to the low cell population and insufficient incubation time, no difference in T-cell number was observed. Nevertheless, the increased number and enhanced immunosuppressive activity of PMN-MDSCs in the Mir4435-2hg−/− group might also suppress T-cell activity, which needs to be further confirmed.

Hypercholesterolemia and dyslipidemia have previously been associated with increased baseline inflammatory responses and higher colorectal cancer incidence (46, 47). Mir4435-2hg depletion only led to high lipid accumulation in MDSCs, with no obvious alteration of plasma cholesterol and triglycerides (Supplementary Fig. S4B) in our mouse models. Conditional knockout of Mir4435-2hg in mice further confirmed that Mir4435-2hg depletion in neutrophils and PMN-MDSCs, but not in intestinal epithelial cells, promoted colorectal cancer development. All together, these data showed that Mir4435-2hg depletion regulated not only the ratio of neutrophils to PMN-MDSCs, but also the immunosuppressive activity of PMN-MDSCs, which contributed to colorectal cancer initiation and progression. Thus, we call for a rigorous reassessment of the biological characterization and clinical significance of lncRNA MIR4435-2HG.

Myelopoiesis is a structured process in which the common myeloid precursors in the bone marrow differentiate into mature circulating leukocytes. However, a variety of pathological conditions, such as chronic inflammation, autoimmune diseases, and cancer, can induce aberrant myelopoiesis, causing the accumulation of immature myeloid cells that diverge from the standard pathway of differentiation (48). Neutrophil differentiation involves several stages, from hematopoietic progenitor cells, common myeloid progenitors, and granulocyte-macrophage progenitors to myeloblasts, myelocytes, metamyelocytes and band forms, and then mature neutrophils (49). Under pathologic conditions, some immature myeloid cells might expand and convert to PMN-MDSCs (50). Consistent with a previous report (35), we observed a decrease in neutrophils in Mir4435-2hg−/− mice, and many differentially expressed genes regulated by Mir4435-2hg associated with mitosis. It needs to be further investigated whether the decrease in neutrophils in Mir4435-2hg−/− mice could increase conversion to PMN-MDSCs. Classical neutrophils, monocytes, and pathologically activated MDSCs coexist in humans and mice within tumors, and MDSCs accumulate with tumor progression (50). Mouse PMN-MDSCs are defined by the surface markers CD11b+Ly6G+Ly6Clo, similar to classical neutrophils, which makes it difficult to distinguish PMN-MDSCs from neutrophils. Therefore, all neutrophils from tumor-bearing mice have been considered PMN-MDSCs for comparison with neutrophils from tumor-free mice in most studies. CD80, CD115, CD124, CD224, and PD-L1 are believed to be PMN-MDSC markers due to their absence on neutrophils in some experimental models (21, 51). However, these markers are not widely accepted. Veglia and colleagues propose CD14 as a specific marker of PMN-MDSCs to distinguish them from classical neutrophils using scRNA-seq in a pure population of neutrophils (43). In our study, we also observed a higher proportion of CD14+ cells among splenic and tumor-infiltrating neutrophils in Mir4435-2hg−/− mice, which represents a higher infiltration of PMN-MDSCs. However, whether CD14 could become the gold standard to discriminate between PMN-MDSCs and classical neutrophils needs further verification. Tumor-associated neutrophils (TAN) are another, related definition of PMN-MDSCs. These comprise a heterogeneous population that includes N1 TANs with antitumor properties and N2 TANs with suppressive functions (52). N2 TANs have been considered PMN-MDSCs (25, 53), and N1 TANs, as normally activated neutrophils, exert antitumor effects by activating unconventional T cells and exerting direct cytotoxic activity against tumors and antimicrobial activity (54). In this study, the decrease in neutrophils in Mir4435-2hg−/− and Mir4435-2hgflox/flox S100a8-Cre mice might also contributed to cancer progression, especially at early-stage disease.

Henao-Mejia and colleagues first reported that murine Mir4435-2hg (termed myeloid RNA regulator of Bim-induced death) could regulate Bim and the short myeloid cell lifespan (35). They also determined that Mir4435-2hg plays a vital role in viral infection (55), leukemia (56–58), and stress-induced abnormalities and clonal hematopoiesis (59). However, most studies on human MIR4435-2HG have focused on cancer biology. Reports analyzing TCGA data for colorectal cancer (60), hepatocellular carcinoma (61), and pan-cancer (62) have revealed that MIR4435-2HG associates with glycolysis and immune infiltration. Together, these data and our data suggest that these correlations should be attributed to the high expression of MIR4435-2HG in immune cells. High immune infiltration leads to a high expression of MIR4435-2HG in tumor tissues. MIR4435-2HG could activate oxidative phosphorylation in myeloid dendritic cells (DCs) from HIV-1 elite controllers by regulating epigenetic modifications and facilitating the transcription of Regulatory-Associated Protein of MTOR Complex 1 (RPTOR) (63). This emerging evidence implies that MIR4435-2HG could also control other immune cells besides neutrophils and PMN-MDSCs, such as DCs and eosinophils, to regulate tumor progression. According to our data, Mir4435-2hg depletion almost resulted in the absence of eosinophils. Higher eosinophil infiltration is reported to associate with better prognosis in colorectal cancer (64). Therefore, we did not exclude the tumor-suppressive roles of eosinophils, which are worth further exploring.

In conclusion, our study demonstrates the unexpected role of MIR4435-2HG using multiple mouse models. It functioned as a tumor suppressor in the tumor stroma by remodeling the immune microenvironment rather than as an oncogene in tumor cells. The deficiency of MIR4435-2HG increased tumor-infiltrating PMN-MDSCs and enhanced their immunosuppressive potential to promote colorectal cancer development, further illustrating mechanisms behind the pathogenesis of colorectal cancer and providing a potential antitumor immunotherapy target.

No disclosures were reported.

H. Yu: Data curation, software, formal analysis, validation, investigation, visualization, methodology, writing–original draft, writing–review and editing. C. Chen: Data curation, software, formal analysis, visualization, methodology, writing–review and editing. F. Han: Data curation, software, formal analysis, methodology. J. Tang: Formal analysis, visualization, methodology. M. Deng: Investigation. Y. Niu: Investigation, writing–original draft. M. Lai: Conceptualization, resources, data curation, supervision, funding acquisition, project administration. H. Zhang: Conceptualization, resources, data curation, supervision, funding acquisition, writing–original draft, project administration, writing–review and editing.

This work is supported by the National Natural Science Foundation of China (81871937, 81672730, 91859204, 82072629, 82173223), the Natural Science Foundation of Zhejiang Province (LZ21H160001) and CAMS Innovation Fund for Medical Sciences (CIFMS, 2019-I2M-5-044). We are grateful to Rongpan Bai, Zhejiang University School of Medicine, for providing APCmin/+ mice. We thank Ms. Xueping Zhou in the Laboratory Animal Center of Zhejiang University for technical assistance on breeding and management of mice and Ms. Qiong Huang for the technical support by the Core Facilities, Zhejiang University School of Medicine.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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

1.
Mercer
TR
,
Dinger
ME
,
Mattick
JS
.
Long non-coding RNAs: insights into functions
.
Nat Rev Genet
2009
;
10
:
155
9
.
2.
Bhan
A
,
Soleimani
M
,
Mandal
SS
.
Long Noncoding RNA and Cancer: A new paradigm
.
Cancer Res
2017
;
77
:
3965
81
.
3.
Zhou
L
,
Zhu
Y
,
Sun
D
,
Zhang
Q
.
Emerging roles of long non-coding RNAs in the tumor microenvironment
.
Int J Biol Sci
2020
;
16
:
2094
103
.
4.
Siegel
RL
,
Miller
KD
,
Fuchs
HE
,
Jemal
A
.
Cancer statistics, 2021
.
CA Cancer J Clin
2021
;
71
:
7
33
.
5.
Kong
J
,
Sun
W
,
Li
C
,
Wan
L
,
Wang
S
,
Wu
Y
, et al
.
Long non-coding RNA LINC01133 inhibits epithelial–mesenchymal transition and metastasis in colorectal cancer by interacting with SRSF6
.
Cancer Lett
2016
;
380
:
476
84
.
6.
Wang
X
,
Yu
H
,
Sun
W
,
Kong
J
,
Zhang
L
,
Tang
J
, et al
.
The long non-coding RNA CYTOR drives colorectal cancer progression by interacting with NCL and Sam68
.
Mol Cancer
2018
;
17
:
110
.
7.
Nötzold
L
,
Frank
L
,
Gandhi
M
,
Polycarpou-Schwarz
M
,
Groß
M
,
Gunkel
M
, et al
.
The long non-coding RNA LINC00152 is essential for cell cycle progression through mitosis in HeLa cells
.
Sci Rep
2017
;
7
:
2265
.
8.
Yang
M
,
He
X
,
Huang
X
,
Wang
J
,
He
Y
,
Wei
L
.
LncRNA MIR4435-2HG-mediated upregulation of TGF-β1 promotes migration and proliferation of nonsmall cell lung cancer cells
.
Environ Toxicol
2020
;
35
:
582
90
.
9.
Qian
H
,
Chen
L
,
Huang
J
,
Wang
X
,
Ma
S
,
Cui
F
, et al
.
The lncRNA MIR4435-2HG promotes lung cancer progression by activating β-catenin signalling
.
J Mol Med Berl Ger
2018
;
96
:
753
64
.
10.
Shen
X
,
Ding
Y
,
Lu
F
,
Yuan
H
,
Luan
W
.
Long noncoding RNA MIR4435-2HG promotes hepatocellular carcinoma proliferation and metastasis through the miR-22–3p/YWHAZ axis
.
Am J Transl Res
2020
;
12
:
6381
94
.
11.
Kong
Q
,
Liang
C
,
Jin
Y
,
Pan
Y
,
Tong
D
,
Kong
Q
, et al
.
The lncRNA MIR4435-2HG is upregulated in hepatocellular carcinoma and promotes cancer cell proliferation by upregulating miRNA-487a
.
Cell Mol Biol Lett
2019
;
24
:
26
.
12.
Wang
H
,
Wu
M
,
Lu
Y
,
He
K
,
Cai
X
,
Yu
X
, et al
.
LncRNA MIR4435-2HG targets desmoplakin and promotes growth and metastasis of gastric cancer by activating Wnt/β-catenin signaling
.
Aging
2019
;
11
:
6657
73
.
13.
Chen
D
,
Tang
P
,
Wang
Y
,
Wan
F
,
Long
J
,
Zhou
J
, et al
.
Downregulation of long non-coding RNA MR4435-2HG suppresses breast cancer progression via the Wnt/β-catenin signaling pathway
.
Oncol Lett
2021
;
21
:
373
.
14.
Deng
L
,
Chi
Y
,
Liu
L
,
Huang
N
,
Wang
L
,
Wu
J
.
LINC00978 predicts poor prognosis in breast cancer patients
.
Sci Rep
2016
;
6
:
37936
.
15.
Xing
P
,
Wang
Y
,
Zhang
L
,
Ma
C
,
Lu
J
.
Knockdown of lncRNA MIR4435-2HG and ST8SIA1 expression inhibits the proliferation, invasion and migration of prostate cancer cells in vitro and in vivo by blocking the activation of the FAK/AKT/β-catenin signaling pathway
.
Int J Mol Med
2021
;
47
:
93
.
16.
Zhang
H
,
Meng
H
,
Huang
X
,
Tong
W
,
Liang
X
,
Li
J
, et al
.
lncRNA MIR4435-2HG promotes cancer cell migration and invasion in prostate carcinoma by upregulating TGF-β1
.
Oncol Lett
2019
;
18
:
4016
21
.
17.
Ouyang
W
,
Ren
L
,
Liu
G
,
Chi
X
,
Wei
H
.
LncRNA MIR4435-2HG predicts poor prognosis in patients with colorectal cancer
.
PeerJ
2019
;
7
:
e6683
.
18.
Shen
M-Y
,
Zhou
G-R
,
Zhang
Z-Y
.
LncRNA MIR4435-2HG contributes into colorectal cancer development and predicts poor prognosis
.
Eur Rev Med Pharmacol Sci
2020
;
24
:
1771
7
.
19.
Dong
X
,
Yang
Z
,
Yang
H
,
Li
D
,
Qiu
X
.
Long Non-coding RNA MIR4435-2HG Promotes Colorectal Cancer Proliferation and Metastasis Through miR-206/YAP1 Axis
.
Front Oncol
2020
;
10
:
160
.
20.
Gabrilovich
DI
,
Bronte
V
,
Chen
S-H
,
Colombo
MP
,
Ochoa
A
,
Ostrand-Rosenberg
S
, et al
.
The terminology issue for myeloid-derived suppressor cells
.
Cancer Res
2007
;
67
:
425
.
21.
Youn
J-I
,
Nagaraj
S
,
Collazo
M
,
Gabrilovich
DI
.
Subsets of Myeloid-Derived Suppressor Cells in Tumor-Bearing Mice
.
J Immunol
2008
;
181
:
5791
802
.
22.
Youn
J-I
,
Kumar
V
,
Collazo
M
,
Nefedova
Y
,
Condamine
T
,
Cheng
P
, et al
.
Epigenetic silencing of retinoblastoma gene regulates pathologic differentiation of myeloid cells in cancer
.
Nat Immunol
2013
;
14
:
211
20
.
23.
Messmer
MN
,
Netherby
CS
,
Banik
D
,
Abrams
SI
.
Tumor-induced myeloid dysfunction and its implications for cancer immunotherapy
.
Cancer Immunol Immunother
2015
;
64
:
1
13
.
24.
Raber
P
,
Ochoa
AC
,
Rodríguez
PC
.
Metabolism of L-Arginine by Myeloid-Derived Suppressor Cells in Cancer: Mechanisms of T cell suppression and Therapeutic Perspectives
.
Immunol Invest
2012
;
41
:
614
34
.
25.
Raber
PL
,
Thevenot
P
,
Sierra
R
,
Wyczechowska
D
,
Halle
D
,
Ramirez
ME
, et al
.
Subpopulations of myeloid-derived suppressor cells impair T cell responses through independent nitric oxide-related pathways
.
Int J Cancer
2014
;
134
:
2853
64
.
26.
Gabrilovich
DI
,
Nagaraj
S
.
Myeloid-derived suppressor cells as regulators of the immune system
.
Nat Rev Immunol
2009
;
9
:
162
74
.
27.
Limagne
E
,
Euvrard
R
,
Thibaudin
M
,
Rébé
C
,
Derangère
V
,
Chevriaux
A
, et al
.
Accumulation of MDSC and Th17 cells in patients with metastatic colorectal cancer predicts the efficacy of a FOLFOX-bevacizumab drug treatment regimen
.
Cancer Res
2016
;
76
:
5241
52
.
28.
OuYang
L-Y
,
Wu
X-J
,
Ye
S-B
,
Zhang
R-X
,
Li
Z-L
,
Liao
W
, et al
.
Tumor-induced myeloid-derived suppressor cells promote tumor progression through oxidative metabolism in human colorectal cancer
.
J Transl Med
2015
;
13
:
47
.
29.
Zheng
Y
,
Tian
X
,
Wang
T
,
Xia
X
,
Cao
F
,
Tian
J
, et al
.
Long noncoding RNA Pvt1 regulates the immunosuppression activity of granulocytic myeloid-derived suppressor cells in tumor-bearing mice
.
Mol Cancer
2019
;
18
:
61
.
30.
Gao
Y
,
Sun
W
,
Shang
W
,
Li
Y
,
Zhang
D
,
Wang
T
, et al
.
Lnc-C/EBPβ negatively regulates the suppressive function of myeloid-derived suppressor cells
.
Cancer Immunol Res
2018
;
6
:
1352
63
.
31.
Gao
Y
,
Wang
T
,
Li
Y
,
Zhang
Y
,
Yang
R
.
Lnc-chop promotes immunosuppressive function of Myeloid-derived suppressor cells in tumor and inflammatory environments
.
J Immunol
2018
;
200
:
2603
14
.
32.
Han
F
,
Zhang
L
,
Chen
C
,
Wang
Y
,
Zhang
Y
,
Qian
L
, et al
.
GLTSCR1 negatively regulates BRD4-dependent transcription elongation and inhibits CRC metastasis
.
Adv Sci
2019
;
6
:
1901114
.
33.
Yoshihara
K
,
Shahmoradgoli
M
,
Martínez
E
,
Vegesna
R
,
Kim
H
,
Torres-Garcia
W
, et al
.
Inferring tumour purity and stromal and immune cell admixture from expression data
.
Nat Commun
2013
;
4
:
2612
.
34.
Trapnell
C
,
Hendrickson
DG
,
Sauvageau
M
,
Goff
L
,
Rinn
JL
,
Pachter
L
.
Differential analysis of gene regulation at transcript resolution with RNA-seq
.
Nat Biotechnol
2013
;
31
:
46
53
.
35.
Kotzin
JJ
,
Spencer
SP
,
McCright
SJ
,
Kumar
DBU
,
Collet
MA
,
Mowel
WK
, et al
.
The long non-coding RNA Morrbid regulates Bim and short-lived myeloid cell lifespan
.
Nature
2016
;
537
:
239
43
.
36.
Neufert
C
,
Becker
C
,
Neurath
MF
.
An inducible mouse model of colon carcinogenesis for the analysis of sporadic and inflammation-driven tumor progression
.
Nat Protoc
2007
;
2
:
1998
2004
.
37.
Robertis
MD
,
Massi
E
,
Poeta
ML
,
Carotti
S
,
Morini
S
,
Cecchetelli
L
, et al
.
The AOM/DSS murine model for the study of colon carcinogenesis: From pathways to diagnosis and therapy studies
.
J Carcinog
2011
;
10
:
9
.
38.
Su
LK
,
Kinzler
KW
,
Vogelstein
B
,
Preisinger
AC
,
Moser
AR
,
Luongo
C
, et al
.
Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene
.
Science
1992
;
256
:
668
70
.
39.
Kuwabara
WMT
,
Andrade-Silva
J
,
Pereira
JNB
,
Scialfa
JH
,
Cipolla-Neto
J
.
Neutrophil activation causes tumor regression in Walker 256 tumor-bearing rats
.
Sci Rep
2019
;
9
:
16524
.
40.
Mishalian
I
,
Granot
Z
,
Fridlender
ZG
.
The diversity of circulating neutrophils in cancer
.
Immunobiology
2017
;
222
:
82
8
.
41.
Ng
LG
,
Ostuni
R
,
Hidalgo
A
.
Heterogeneity of neutrophils
.
Nat Rev Immunol
2019
;
19
:
255
65
.
42.
Urdinguio
RG
,
Fernandez
AF
,
Moncada-Pazos
A
,
Huidobro
C
,
Rodriguez
RM
,
Ferrero
C
, et al
.
Immune-dependent and independent antitumor activity of GM-CSF aberrantly expressed by mouse and human colorectal tumors
.
Cancer Res
2013
;
73
:
395
405
.
43.
Veglia
F
,
Hashimoto
A
,
Dweep
H
,
Sanseviero
E
,
De Leo
A
,
Tcyganov
E
, et al
.
Analysis of classical neutrophils and polymorphonuclear myeloid-derived suppressor cells in cancer patients and tumor-bearing mice
.
J Exp Med
2021
;
218
:
e20201803
.
44.
Veglia
F
,
Tyurin
VA
,
Blasi
M
,
De Leo
A
,
Kossenkov
AV
,
Donthireddy
L
, et al
.
Fatty acid transport protein 2 reprograms neutrophils in cancer
.
Nature
2019
;
569
:
73
8
.
45.
Hicks
KC
,
Tyurina
YY
,
Kagan
VE
,
Gabrilovich
DI
.
Myeloid Cell–Derived Oxidized Lipids and Regulation of the Tumor Microenvironment
.
Cancer Res
2022
;
82
:
187
94
.
46.
Tall
AR
,
Yvan-Charvet
L
.
Cholesterol, inflammation and innate immunity
.
Nat Rev Immunol
2015
;
15
:
104
16
.
47.
Cowey
S
,
Hardy
RW
.
The Metabolic Syndrome
.
Am J Pathol
2006
;
169
:
1505
22
.
48.
Bronte
V
,
Brandau
S
,
Chen
S-H
,
Colombo
MP
,
Frey
AB
,
Greten
TF
, et al
.
Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards
.
Nat Commun
2016
;
7
:
12150
.
49.
Evrard
M
,
Kwok
IWH
,
Chong
SZ
,
Teng
KWW
,
Becht
E
,
Chen
J
, et al
.
Developmental analysis of bone marrow neutrophils reveals populations specialized in expansion, trafficking, and effector functions
.
Immunity
2018
;
48
:
364
79
.
50.
Veglia
F
,
Perego
M
,
Gabrilovich
D
.
Myeloid-derived suppressor cells coming of age
.
Nat Immunol
2018
;
19
:
108
19
.
51.
Youn
J-I
,
Collazo
M
,
Shalova
IN
,
Biswas
SK
,
Gabrilovich
DI
.
Characterization of the nature of granulocytic myeloid-derived suppressor cells in tumor-bearing mice
.
J Leukoc Biol
2012
;
91
:
167
81
.
52.
Fridlender
ZG
,
Sun
J
,
Kim
S
,
Kapoor
V
,
Cheng
G
,
Ling
L
, et al
.
Polarization of tumor-associated neutrophil phenotype by TGF-beta: “N1” versus “N2” TAN
.
Cancer Cell
2009
;
16
:
183
94
.
53.
Cimen Bozkus
C
,
Elzey
BD
,
Crist
SA
,
Ellies
LG
,
Ratliff
TL
.
Expression of cationic amino acid transporter 2 is required for Myeloid-derived suppressor Cell-mediated control of T cell immunity
.
J Immunol
2015
;
195
:
5237
50
.
54.
Jaillon
S
,
Ponzetta
A
,
Di Mitri
D
,
Santoni
A
,
Bonecchi
R
,
Mantovani
A
.
Neutrophil diversity and plasticity in tumour progression and therapy
.
Nat Rev Cancer
2020
;
20
:
485
503
.
55.
Kotzin
JJ
,
Iseka
F
,
Wright
J
,
Basavappa
MG
,
Clark
ML
,
Ali
M-A
, et al
.
The long noncoding RNA Morrbid regulates CD8 T cells in response to viral infection
.
Proc Natl Acad Sci U S A
2019
;
116
:
11916
25
.
56.
Cai
Z
,
Aguilera
F
,
Ramdas
B
,
Daulatabad
SV
,
Srivastava
R
,
Kotzin
JJ
, et al
.
Targeting Bim via a lncRNA morrbid regulates the survival of preleukemic and leukemic cells
.
Cell Rep
2020
;
31
:
107816
.
57.
Cai
Z
,
Lu
X
,
Zhang
C
,
Nelanuthala
S
,
Aguilera
F
,
Hadley
A
, et al
.
Hyperglycemia cooperates with Tet2 heterozygosity to induce leukemia driven by proinflammatory cytokine-induced lncRNA Morrbid
.
J Clin Invest
2021
;
131
:
140707
.
58.
Cai
Z
,
Zhang
C
,
Kotzin
JJ
,
Williams
A
,
Henao-Mejia
J
,
Kapur
R
.
Role of lncRNA Morrbid in PTPN11(Shp2)E76K-driven juvenile myelomonocytic leukemia
.
Blood Adv
2020
;
4
:
3246
51
.
59.
Cai
Z
,
Kotzin
JJ
,
Ramdas
B
,
Chen
S
,
Nelanuthala
S
,
Palam
LR
, et al
.
Inhibition of inflammatory signaling in Tet2 mutant preleukemic cells mitigates Stress-induced abnormalities and clonal hematopoiesis
.
Cell Stem Cell
2018
;
23
:
833
49
.
60.
Chen
J
,
Song
Y
,
Li
M
,
Zhang
Y
,
Lin
T
,
Sun
J
, et al
.
Comprehensive analysis of ceRNA networks reveals prognostic lncRNAs related to immune infiltration in colorectal cancer
.
BMC Cancer
2021
;
21
:
255
.
61.
Bai
Y
,
Lin
H
,
Chen
J
,
Wu
Y
,
Yu
S
.
Identification of prognostic Glycolysis-related lncRNA signature in tumor immune microenvironment of hepatocellular carcinoma
.
Front Mol Biosci
2021
;
8
:
645084
.
62.
Ho
K-H
,
Huang
T-W
,
Shih
C-M
,
Lee
Y-T
,
Liu
A-J
,
Chen
P-H
, et al
.
Glycolysis-associated lncRNAs identify a subgroup of cancer patients with poor prognoses and a high-infiltration immune microenvironment
.
BMC Med
2021
;
19
:
59
.
63.
Hartana
CA
,
Rassadkina
Y
,
Gao
C
,
Martin-Gayo
E
,
Walker
BD
,
Lichterfeld
M
, et al
.
Long noncoding RNA MIR4435-2HG enhances metabolic function of myeloid dendritic cells from HIV-1 elite controllers
.
J Clin Invest
2021
;
131
:
e146136
.
64.
Prizment
AE
,
Vierkant
RA
,
Smyrk
TC
,
Tillmans
LS
,
Lee
JJ
,
Sriramarao
P
, et al
.
Tumor eosinophil infiltration and improved survival of colorectal cancer patients: Iowa Women's Health Study
.
Mod Pathol
2016
;
29
:
516
27
.

Supplementary data