Open Access

Determination of long non‑coding RNAs associated with EZH2 in neuroblastoma by RIP‑seq, RNA‑seq and ChIP‑seq

  • Authors:
    • Mujie Ye
    • Lulu Xie
    • Jingjing Zhang
    • Baihui Liu
    • Xiangqi Liu
    • Jiajun He
    • Duan Ma
    • Kuiran Dong
  • View Affiliations

  • Published online on: July 13, 2020
  • Article Number: 1
  • Copyright: © Ye et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Neuroblastoma (NB) is the most common type of extracranial solid tumor found in children. Despite several treatment options, patients with advanced stage disease have a poor prognosis. Previous studies have reported that enhancer of zeste homolog 2 (EZH2) and long non‑coding RNAs (lncRNAs) have abnormal expression levels in NB and participate in tumorigenesis and NB development. However, the association between EZH2 and lncRNAs remain unclear. In the present study, RNA immunoprecipitation‑sequencing (RIP‑seq) was used to analyze the lncRNAs binding to EZH2. Following EZH2 knockdown via short hairpin RNA, RNA‑seq was performed in shEZH2 and control groups in SH‑SY5Y cells. Chromatin IP (ChIP)‑seq was used to determine the genes that may be regulated by EZH2. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to identify the signaling pathways involved in NB. The results from RIP‑seq identified 94 lncRNAs, including SNHG7, SNHG22, KTN‑AS1 and Linc00843. Furthermore, results from RNA‑seq demonstrated that, following EZH2 knockdown, 448 genes were up‑ and 571 genes were downregulated, with 32 lncRNAs up‑ and 35 downregulated and differentially expressed compared with control groups. Certain lncRNAs, including MALAT1, H19, Linc01021 and SNHG5, were differentially expressed in EZH2‑knockdown group compared with the control group. ChIP‑seq identified EZH2 located in the promoter region of 138 lncRNAs including CASC16, CASC15, LINC00694 and TBX5‑AS1. In summary, the present study demonstrated that certain lncRNAs directly bound EZH2 and regulated EZH2 expression levels. A number of these lncRNAs that are associated with EZH2 may participate in NB tumorigenesis.


Neuroblastoma (NB) is an embryonic tumor derived from sympathetic neural crest cells (1,2). It is the most common type of extracranial solid tumor found in children and the most common type of malignant tumor found in infants and young children, with an incidence rate of 10.5 per million children younger than 14 years old (35). The site of the disease is concealed, and NB easily metastasizes (5). Low-risk NB has the characteristics of regression and inducing NB differentiation and maturation in vitro, while high-risk NB has high malignancy and can metastasize early (6). The prognosis of patients with NB is extremely poor, which accounts for ~12–15% of all pediatric cancer-associated deaths (7,8). In recent years, treatments for low- and medium-risk NB have improved; however, the cure rate for patients with high-risk NB remains low (9,10). It is therefore crucial to develop novel treatments for patients with high-risk NB.

Enhancer of zeste homolog 2 (EZH2) protein is the core catalytic element of polycomb repressor complex 2 (PRC2), which regulates the transcription of target genes via promoting histone H3 methylation (1113). Previous studies have demonstrated that high expression level of EZH2 is a poor prognostic factor in various types of cancer (14,15). For example, in pancreatic cancer, EZH2-mediated microRNA-139-5p regulates epithelial-mesenchymal transition and lymph node metastasis (16). Moreover, EZH2 and EED directly regulate androgen receptor in advanced prostate cancer (17). In NB, EZH2 is highly expressed and can epigenetically silences NB tumor suppressor genes, including CASZ1, CLU, RUNX3 and NGFR, suggesting that EZH2 may be an NB molecular target (18).

Long non-coding RNAs (lncRNAs) are a type of RNA of >200 nucleotides in length that do not encode for proteins. Following their discovery, lncRNAs were considered as ‘noises’ in the genome transcription process (19,20). However, subsequent studies demonstrated that lncRNAs regulate genes at a number of levels, including epigenetic, genomic transcription and post-transcriptional levels. lncRNAs participate in cancer development and tumorigenesis by affecting tumor cell proliferation, migration, invasion, apoptosis and the cell cycle, and promoting angiogenesis (21,22). lncRNAs directly bind to EZH2, recruiting it to the promoter region of genes to repress their expression levels. lncRNAs also serve as EZH2 effectors or regulators (2325). The present study investigated the association between lncRNAs and EZH2 using RNA immunoprecipitation (RIP)-, RNA- and chromatin IP-sequencing (ChIP-seq).

Materials and methods

Cell culture

The SH-SY5Y cell line was obtained from the American Type Culture Collection (cat. no. CRL-2266) and the 293T cell line was obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. Cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% FBS and 1% penicillin-streptomycin solution (all from Biological Industries) and placed in a humidified incubator with 5% CO2 at 37°C. Cell culture dishes were obtained from Hangzhou Xinyou Biotechnology Co., Ltd.

Construction of short hairpin (sh)RNAs and stable transfected cell lines

EZH2 shRNA plasmids [designed online (] and pLKO1(Genomeditech, Shanghai, China) were selected as vectors. The shRNA target sequences were as follows: Forward shEZH2-F, 5′-CCGGCCCAACATAGATGGACCAAATCTCGAGATTTGGTCCATCTATGTTGGGTTTTTG-3′; reverse shEZH2-R, 5′AATTCAAAAACCCAACATAGATGGACCAAATCTCGAGATTTGGTCCATCTATGTTGGG-3′; forward shControl-F, 5′-CCGGTTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAATTTTTG-3′; reverse shControl-R, 5′-AATTCAAAAATTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAA. Lentivirus with shEZH2 was packaged with 293T cells using transfection reagent Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Stably transfected SH-SY5Y cells with shRNA (12 µg) and control (12 µg) were acquired following puromycin selection.

Western blotting

Cell were lysed using RIPA lysis buffer (Beyotime Institute of Biotechnology) on ice, and western blotting was performed as standard (26). Following blocking with 8% skimmed milk for 1 h at room temperature, membranes were incubated with primary antibodies (EZH2; cat. no. 5246S; and β-tubulin; cat. no. 2146S; Cell Signaling Technology, Inc.) on a decolorization shaker at 4°C overnight. After washing with Tris-HCl + 0.05% Tween-20 TBST three times, membranes were incubated with secondary antibody (goat anti-rabbit IgG; 1:2,000; cat. no. CW0107; CoWin Biosciences) for 1 h at room temperature. Protein signals were detected via enhanced chemiluminescence substrate (EMD Millipore).


RNA Immunoprecipitation (RIP) was performed followed as Gagliardi et al (27) In simple terms, RNA was enriched with EZH2 antibody (1:100; cat. no. 5246S; Cell Signaling Technology, Inc) in SH-SY5Y cells. Enriched RNA was broken into short fragments using fragmentation buffer (cat. no. N402-VAHTS; Vazyme Biotech Co., Ltd.) at 94°C for 5 min. Fragmented mRNA was used as a template to synthesize cDNA using random hexamers, buffer, dNTPs and DNA polymerase I (VAHTS Stranded mRNA-seq Library Prep kit for Illumina; cat. no. NR602-01; Vazyme Biotech Co., Ltd.). After the synthesis of the double-stranded cDNA, the double-stranded cDNA was purified. LC magnetic beads were used for purification and target fragment binding. The EP tube was placed on a magnetic stand (beads combined with cDNA), then the supernatant was removed, and washed twice with 80% ethanol for 30 sec each time. End-repair and library preparation was performed by the aforementioned kit (VAHTS Stranded mRNA-seq Library Prep kit for Illumina). Target size selection was performed together with magnetic purification. PCR amplification (using Amplification Mix; cat. no. N611-01; Vazyme Biotech Co., Ltd.) was then performed as following: Initial denaturation 95°C for 3 min, 12 cycles of denaturation at 98°C for 20 sec, annealing at 55°C for 15 sec, elongation at 72°C for 30 sec, and final extension 72°C for 5 min. The primer sequences of PCR were as follows: Forward, 5′AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′; reverse, 5′-CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3′. Agarose electrophoresis was used for quality inspection of the constructed library. Qubit 2.0 (Invitrogen; Thermo Fisher Scientific, Inc.) was used to detect the concentration of the library, and the loading concentration of the library was 42 ng/µl with 20 µl. After library quality tests were passed, libraries were sequenced using an Novaseq 6000 sequencer (Illumina, Inc.) with PE150 model (double-ended 150 bp sequencing) according to effective concentration and target data volume. RIP-seq and subsequent bioinformatics Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed by Hangzhou Lianchuan Biotechnology Co., Ltd with the DAVID website (

RNA-seq and analysis

EZH2 protein was knocked down in SH-SY5Y cells using shRNA. Total RNA was obtained from EZH2 knockdown and control groups with TRIzol® (Takara Bio, Inc.). Each group included three replicates. Illumina Paired End Sample Prep kits (Illumina, Inc.) were used to prepare libraries. Each cDNA library was sequenced using an Illumina Hiseq 4000 (cat. no. PE150; Illumina, Inc.). Differential expression levels of lncRNA and mRNA transcripts between the EZH2 knockdown and control groups were measured. RNA-seq and subsequent GO and KEGG analysis was performed by Hangzhou Lianchuan Biotechnology Co., Ltd following the previous study (28).


Cells from one 10-cm dish of 80–90% confluence cultures were sonicated 4 times for (30 sec on and 30 sec off) in precooled conditions (Fisher Sonic Dismembrator; Thermo Fisher Scientific, Inc.). DNA was disrupted into fragments of 200–1,000 bp by nucleic acid gel. Anti-EZH2 (1:100; cat. no. 5246S; Cell Signaling Technology, Inc) was used to capture chromatin fragments from cell extracts, and libraries were constructed from immunoprecipitated DNA. An Illumina sequencer was used for high-throughput sequencing of lncRNA and mRNA. ChIP-seq and subsequent GO and KEGG analysis were performed by Hangzhou Lianchuan Biotechnology Co., Ltd. The ChIP protocol was conducted as previously described by Kong et al (29).

Differential expression level analysis

Gene expression levels were estimated using fragments per kilobase of transcript per million mapped reads (FPKM) values. Cuffdiff (v2.1.1; was used to calculate FPKM values of lncRNAs and mRNAs.DAVID ( was used to perform GO and KEGG analysis. Official gene symbols of the significantly different genes were enriched. We followed the instructions on the website step by step until acquiring GO and KEGG terms. Significantly differentially expressed genes were obtained using P-value <0.05.


RIP-seq identifies EZH2-interacting lncRNAs

RNA Immunoprecipitation (RIP) was performed using SH-SY5Y cells and western blotting was used to check IP efficiency (Fig. 1A). Both IP supernatants and inputs showed bands for the internal reference gene GAPDH, demonstrating protein extraction and the western blotting system is successful. Antibody heavy chain was detected in the IP and IgG lanes, indicating that IP was successful. Distribution statistics for peaks of each functional area of the gene demonstrated that the coding region accounted for 50% and the exon region of a non-coding gene was ~20% (Fig. 1B). By analyzing the distribution of RPKM values, the gene expression level characteristics of the sample were treated as a whole. If IP was significantly enriched compared with the input group, the total expression level of all genes in the IP group should have been higher than that in the input group (Fig. 1C). lncRNAs are >200 nucleotides in length. Length distribution statistics of known lncRNAs were determined. The results demonstrated that lncRNAs 500–1,000 and >3,500 bp in length were significantly more common than lncRNAs of other lengths in SH-SY5Y cells (Fig. 1D). A total of 2,595 lncRNAs were identified by counting peaks associated with known lncRNAs. Among these lncRNAs, 94 were identified via exclusion of processed transcripts and retained introns (Table I). GO analysis demonstrated that these lncRNAs were involved in numerous biological processes, including cellular components and molecular functions, such as metabolic process and transcription regulation(Fig. 1E). Furthermore, KEGG analysis indicated that ‘Hedgehog signaling pathway’ and ‘FoxO signaling pathway’ were enriched in SH-SY5Y cells. ‘Tight junction’, ‘apoptosis’ and ‘cell cycle’ may also be associated with these lncRNAs (Fig. 1F).

Table I.

lncRNAs determined by RNA immunoprecipitation-sequencing.

Table I.

lncRNAs determined by RNA immunoprecipitation-sequencing.

lncRNA namelncRNA IDlncRNA typeChromosome
AF001548.2-201 ENST00000574212antisense_RNA16
AC087280.2-201 ENST00000637205lincRNA11
HOXA11-AS-205 ENST00000522863antisense_RNA7
PCBP1-AS1-210 ENST00000416395antisense_RNA2
AP002383.3-201 ENST00000545958antisense_RNA11
CCDC18-AS1-205 ENST00000421202lincRNA1
AL096701.3-202 ENST00000483736antisense_RNA22
AC020661.4-201 ENST00000561388antisense_RNA15
PITRM1-AS1-201 ENST00000430356antisense_RNA10
AL121759.1-201 ENST00000434043antisense_RNA20
AC244517.2-201 ENST00000607216antisense_RNA5
PPP1R26-AS1-202 ENST00000603624antisense_RNA9
AC025188.1-201 ENST00000507514antisense_RNA5
AC092115.3-201 ENST00000575838antisense_RNA16
RAB11B-AS1-201 ENST00000593581antisense_RNA19
AC069503.1-201 ENST00000538710lincRNA12
LINC00843-201 ENST00000429104lincRNA10
AC011477.3-201 ENST00000585571antisense_RNA19
AC010320.4-201 ENST00000594379antisense_RNA19
AL135787.1-201 ENST00000450154antisense_RNA9
AC109587.1-201 ENST00000482368antisense_RNA3
FAM201A-201 ENST00000377680antisense_RNA9
SNHG7-201 ENST00000414282antisense_RNA9
AC138150.1-201 ENST00000589950antisense_RNA17
EMX2OS-206 ENST00000551288antisense_RNA10
AC137932.1-201 ENST00000563087antisense_RNA16
AC005920.2-201 ENST00000509833antisense_RNA17
MZF1-AS1-202 ENST00000600534antisense_RNA19
AC080112.2-201 ENST00000578774antisense_RNA17
AC008741.1-201 ENST00000569456antisense_RNA16
AC024563.1-201 ENST00000601860antisense_RNA19
AC020911.1-201 ENST00000591038antisense_RNA19
AC097467.3-216 ENST00000599555antisense_RNA4
C5orf66-205 ENST00000555438antisense_RNA5
SGO1-AS1-204 ENST00000634618lincRNA3
AC011603.3-201 ENST00000549516antisense_RNA12
ADIRF-AS1-203 ENST00000609111antisense_RNA10
AC106864.1-201 ENST00000510655antisense_RNA4
AC093110.1-202 ENST00000626206antisense_RNA2
MCM3AP-AS1-201 ENST00000414659antisense_RNA21
AC108449.1-202 ENST00000517632antisense_RNA8
AC004923.4-201 ENST00000532296antisense_RNA11
KDM4A-AS1-203 ENST00000434346antisense_RNA1
FO393401.1-201 ENST00000453914antisense_RNA20
AC078777.1-201 ENST00000425371antisense_RNA12
PDZRN3-AS1-201 ENST00000478988antisense_RNA3
AL139423.1-201 ENST00000606802antisense_RNA1
AL118558.1-201 ENST00000557551antisense_RNA14
FOXD1-AS1-201 ENST00000514661lincRNA5
ZFPM2-AS1-207 ENST00000524045antisense_RNA8
AC073167.1-201 ENST00000559303antisense_RNA15
SH3BP5-AS1-202 ENST00000420195antisense_RNA3
AC012170.2-201 ENST00000560380antisense_RNA15
NEXN-AS1-201 ENST00000421331antisense_RNA1
AC092329.1-201 ENST00000594653lincRNA19
DNMBP-AS1-202 ENST00000434409antisense_RNA10
AC013391.1-201 ENST00000560477antisense_RNA15
PKP4-AS1-201 ENST00000342892antisense_RNA2
LINC01089-202 ENST00000429892lincRNA12
AC010978.1-202 ENST00000427050antisense_RNA2
AL606534.3-201 ENST00000437499antisense_RNA1
AL606534.1-201 ENST00000439562antisense_RNA1
AL356599.1-205 ENST00000606388antisense_RNA6
KTN1-AS1-202 ENST00000412224antisense_RNA14
AC004893.2-201 ENST00000360902antisense_RNA7
AC087286.3-201 ENST00000561409antisense_RNA15
AL139021.2-201 ENST00000556390antisense_RNA14
C1orf220-202 ENST00000521244lincRNA1
H1FX-AS1-201 ENST00000383461antisense_RNA3
SEC62-AS1-201 ENST00000479626antisense_RNA3
AL161747.2-201 ENST00000535893antisense_RNA14
DDN-AS1-202 ENST00000547866antisense_RNA12
TTC3-AS1-201 ENST00000424733antisense_RNA1
AC245452.1-201 ENST00000458178antisense_RNA22
AC008676.1-201 ENST00000508443antisense_RNA5
AC022395.1-201 ENST00000451610antisense_RNA10
AC010976.1-203 ENST00000629005antisense_RNA2
AP000229.1-201 ENST00000608591lincRNA21
AC026427.1-201 ENST00000508993antisense_RNA5
AC025043.1-201 ENST00000558047antisense_RNA15
SLC16A1-AS1-204 ENST00000428411antisense_RNA1
ZNF337-AS1-201 ENST00000414393antisense_RNA20
AL451047.1-201 ENST00000424451antisense_RNA1
AC009185.1-201 ENST00000517634antisense_RNA5
AC023790.2-201 ENST00000543321lincRNA12
AC092143.3-201 ENST00000565150antisense_RNA16
ASH1L-AS1-202 ENST00000456633antisense_RNA1
SPG20-AS1-203 ENST00000493739antisense_RNA13
ALKBH3-AS1-201 ENST00000499194antisense_RNA11
SNHG22-201 ENST00000589499antisense_RNA18
AC010624.3-201 ENST00000599914antisense_RNA19
AL592166.1-202 ENST00000428791antisense_RNA1

[i] lncRNA, long non-coding RNA; lincRNA, large intergenic noncoding RNA.

RNA-seq for mRNAs and lncRNAs

The results from western blotting demonstrated that EZH2 was significantly downregulated following shRNA transfection (Fig. 2A). By analyzing the distribution of FPKM values, the gene expression level characteristics of the sample were treated as a whole (Fig. 2B). Following EZH2 knockdown, 448 up- and 571 downregulated genes were differentially expressed compared with the normal control group (Fig. 2C and D). A heatmap of the top 100 differentially expressed genes was generated (Fig. 2E). GO analysis demonstrated that these genes were associated with ‘negative regulation of neuron apoptotic processes’, ‘nervous system development’ and ‘peripheral nervous system development’. KEGG analysis showed that enriched genes were primarily distributed in the ‘TGF-β signaling pathway’, ‘Hippo signaling pathway’ and ‘cAMP signaling pathway’. Compared with the normal control group, 32 up- and 35 downregulated lncRNAs were differentially expressed in the shEZH2 group (Fig. 3A and B; Table II). A heatmap of the top 100 differentially expressed lncRNAs (including known and novel lncRNAs) was generated (Fig. 3C). GO analysis demonstrated that these lncRNAs were involved in numerous biological processes, including ‘regulation of developmental growth’, ‘peptidyl-tyrosine phosphorylation’ and ‘histone glutamine methylation’ (Fig. 3D). KEGG analysis demonstrated that ‘Hedgehog signaling pathway’, ‘Parkinson's disease’ and ‘Alzheimer's disease’ were associated with these lncRNAs (Fig. 3E).

Table II.

Different lncRNAs determined by RNA-sequencing following enhancer of zeste homolog 2 knockdown.

Table II.

Different lncRNAs determined by RNA-sequencing following enhancer of zeste homolog 2 knockdown.

lncRNA namelncRNA IDChromosomeRegulation
MALAT1 ENST0000061813211Down
AL360012.1 ENST000006028131Down
TTN-AS1 ENST000006291172Down
AC108488.1 ENST000004229612Up
EXOC3-AS1 ENST000006236735Down
AC015813.1 ENST0000058209617Up
AC025171.2 ENST000005151085Down
DHRS4-AS1 ENST0000055504514Down
AC027237.3 ENST0000055861715Down
SVIL-AS1 ENST0000042706310Down
SNHG15 ENST000004387057Down
SNHG5 ENST000004310436Up
AC125494.1 ENST0000039679912Down
AC079781.5 ENST000006413907Down
LINC00958 ENST0000053447711Up
LRRC75A-AS1 ENST0000047236717Down
H19 ENST0000041278811Up
FAM212B-AS1 ENST000004303731Up
AC016757.1 ENST000004099422Up
AL590133.2 ENST000005604811Down
THAP7-AS1 ENST0000042996222Up
EBLN3P ENST000006289249Down

[i] lncRNA, long non-coding RNA.

ChIP-seq for EZH2

Due to the influence of chromosome conformation, chromosome expression levels in the active region of gene expression levels was more open. This resulted in input DNA reads exhibiting greater abundance in promoter and gene body regions, with a characteristic decrease near the transcription start site (TSS). The distribution of IP DNA is associated with EZH2 proteins, and apparent modifications such as transcription factors and H3K27me3 were enriched in the promoter and gene body regions. Through the distribution of reads in the intervals of these genes, the success of ChIP-seq experiments was verified (Fig. 4B). Analysis of peak distribution in the genomic functional area indicated that intergenic and promoter-TSS were the most frequent areas (Fig. 4A). ChIP-seq identified 634 genes located in the promoter region, including 138 long intervening non-coding RNAs (lincRNA; Table III). GO analysis demonstrated enrichment of ‘nervous system development’, ‘chemical synaptic transmission’ and ‘trans-synaptic signaling’ (Fig. 4C). KEGG analysis indicated that ‘Rap1 signaling pathway’, ‘cAMP signaling pathway’ and ‘retrograde endocannabinoid signaling’ were enriched (Fig. 4D).

Table III.

Promoter of long non-coding RNA determined by chromatin immunoprecipitation-sequencing.

Table III.

Promoter of long non-coding RNA determined by chromatin immunoprecipitation-sequencing.

Gene IDChromosomeStartEndStrand Annotated_TranscriptlogFCP-value
RP11-672L10.318907879908507 ENST000005825543.129033173 1.33492×10−12
KIRREL311127003233127004161 ENST000005330263.003238319 1.50761×10−06
KIRREL311127000973127001187 ENST000005477383.003238319 1.50761×10−06
IGF2-AS1121396212141215+ ENST000003813612.877117623 1.14607×10−17
AP000282.2213307094633071173 ENST000004546222.813201993 4.67603×10−05
RP11-88H9.21111989337111991186+ ENST000004382932.775316437 2.86341×10−10
TBX5-AS112114407959114408321+ ENST000005312022.734420323 9.25093×10−07
MIR1-1HG206254983962550053+ ENST000006249142.522145791 9.65857×10−10
FLJ16779206325327763253737+ ENST000006127222.510159699 1.96314×10−09
RP11-92C4.699894299298944517 ENST000006056312.391645807 1.12556×10−08
RP11-672L10.218904551904765 ENST000005829212.372166725 1.71009×1007
RP11-672L10.218904176904390 ENST000005829212.372166725 1.71009×10−07
RP11-672L10.218906549907654 ENST000005817192.372166725 1.71009×10−07
RP3-525N10.266863416668635846 ENST000006043922.366348664 2.47756×10−10
AC099754.132662245126622769 ENST000004358842.28090657 4.94043×10−05
RP11-436F23.144638976346390468+ ENST000005024552.248486391 1.13494×10−08
CTC-235G5.357608415676085290 ENST000005036522.221930293 2.35777×10−09
RP11-274G22.1X2137365821374071 ENST000006363172.211953324 1.02635×10−11
RP11-343J3.2106957470869575542+ ENST000004287532.207584122 1.94744×10−06
AP002856.511131252716131253002+ ENST000004165532.2033608430.001177692
RP11-452C13.17157867952157868291+ ENST000006085962.194013770.001695242
GPR50-AS1X151177247151178372 ENST000004541962.152759945 3.44626×10−06
LINC002001011592591159473+ ENST000004256302.1515204640.000313333
RNF219-AS1137791884677919422+ ENST000006078622.1454101210.000124562
C8orf34-AS186833142568331669 ENST000005122942.1294078060.00016937
RP11-17E2.242194801621948230+ ENST000005107052.1294078060.00016937
RP1-269M15.3204318934943189646+ ENST000006117912.1294078060.00016937
RP6-24A23.3X108735146108736079+ ENST000006088112.103500759 2.23745×10−05
RP4-683L5.1113541815835420102+ ENST000005341652.073937887 6.79163×10−10
PTPRD-AS291061245910613283+ ENST000004295812.046198680.000273436
LINC012103137771274137772805+ ENST000004787722.039638671 5.14222×10−08
RP11-563K23.17143363541143363755+ ENST000006096742.008614264 1.33265×10−05
RP11-588H7.131460156614601780+ ENST000006358001.9897445890.00057344
RP11-343P9.18135456592135457331 ENST000005186741.980487281 5.20702×10−06
FGF14-AS113102368126102368468+ ENST000004516301.9700949040.004327089
CTD-2523D13.211119729856119730070+ ENST000005332531.9546545550.000134587
RP11-901H12.132122712321227337 ENST000006349471.9503339850.006647239
CASC16165260666052606874 ENST000005102381.9438766170.010319298
ADGRA1-AS110133088588133089156 ENST000003660991.898488801 8.76189×10−06
RP11-231C18.145433187854332092+ ENST000005116341.8832833820.001289255
MIR519A1195375193453752148+ ENST000003852571.8744994420.025042572
MIR129-2114358082543581855+ ENST000003622071.872185866 1.61125×10−07
CD1E1158352934158353309+ ENST000003681671.8477659530.00285251
RP11-415C15.142714071327140939 ENST000005068781.824616520.001888542
RP11-21C4.186457810364578381 ENST000005208341.797854867 1.29304×10−05
AL022344.7104275190942752386 ENST000005689761.7961426770.000430003
HAR1B206310260063103193 ENST000004479101.7847417550.000108551
RP1-273N12.469883285898833303 ENST000006354231.7828295440.000111871
RP11-461O7.1165619069756191236 ENST000005012591.7428594380.000158433
MAPT-AS1174589535945895627 ENST000005795991.733234586 9.03861×10−06
RP1-90G24.10223220419932204413+ ENST000004349421.7056846750.000158433
HAR1A206310205963102459+ ENST000004331611.7052575320.000218753
LL22NC03-121E8.3224814148748141701+ ENST000004463641.6996010220.004015236
CTD-2194D22.3518828061883957+ ENST000005063351.67006809 4.45899×10−07
FOXC2-AS1168656760886568108 ENST000005632801.6670994730.000418716
MIR124-2HG86437699664377210+ ENST000005240601.6560918260.009625718
RP11-876N24.2161088869610889194 ENST000005720171.6453685520.001847602
AC010729.1256958775696091+ ENST000004555791.6340910460.000299766
RP11-146I2.161508992715090141 ENST000004376481.6327812490.005823769
CTD-2089N3.155096724250967513 ENST000005103491.6245693790.009038249
RP11-386B13.34185051181185051395+ ENST000005090171.6145000150.009625718
RP11-1263C18.24576322576536+ ENST000006376741.6111319060.001648005
RP11-486P11.172009599620096210+ ENST000005909121.6085599260.014306174
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Although numerous treatments for NB currently exist, patients with NB have only 40% survival rate (2,30). A novel treatment is therefore needed to improve survival rate. EZH2 is a member of the polycomb group protein family that is upregulated in various types of cancer, including NB (31,32). Li et al (32) demonstrated that EZH2 knockdown significantly inhibits NB differentiation. Transcriptome sequencing has demonstrated that neurotrophic receptor tyrosine kinase 1 may be a target of EZH2. Chen et al (31) reported that the MYCN gene binds to the EZH2 promoter, directly promoting EZH2 expression and EZH2 inhibition of neuronal differentiation in a PRC2-dependent manner (33). Tsubota et al (34) demonstrated that EZH2 inhibitors significantly repress the growth of tyrosine hydroxylase-MYCN NB mice, and that MYCN and PRC2 targets are positively correlated in NB. EZH2 may therefore be considered as a novel target for NB treatment. Bate-Eya et al (35) demonstrated that high expression of EZH2 has a survival function independent of its methyltransferase activity in NB. Although inhibitors of EZH2 are at pre-clinical stage in many cancers, their efficacy and underlying mechanism in NB remain unknown.

In a previous study, certain lncRNAs were demonstrated to serve key roles in NB. For example, FOXD3-antisense RNA (AS) 1 is downregulated in NB tissues and cell lines; this is an independent prognostic marker for favorable outcomes for patients with NB. FOXD3-AS1 inhibits the progression of NB via repressing poly-ADP ribose polymerase 1-mediated CCCTC-binding factor activation (36). The lncRNA pancEts-1 is upregulated and is an independent prognostic factor for unfavorable NB outcomes. In addition, pancEts-1 directly interacts with heterogeneous nuclear ribonucleoprotein K to increase its interaction with β-catenin, resulting in stabilization and transactivation of β-catenin and promotion of the growth and metastasis of NB both in vitro and in vivo (37). EZH2 is a transcriptional repressor associated with lncRNA. Numerous lncRNAs are associated with EZH2 with positive or negative correlation (38,39). Since the interacting gene product enhances the co-expressed gene, positively correlated lncRNA is a potential ligand for EZH2 or has the same transcriptional machinery as EZH2 (40). Knocking down EZH2 using small interfering RNA has previously confirmed that lncRNA is negatively correlated with EZH2 expression and is inhibited by EZH2 (41). The present study demonstrated that numerous lncRNAs were associated with EZH2. RIP-seq identified 94 lncRNAs that may bind to EZH2 directly. Among lncRNAs, Chi et al (42) reported that small nucleolar host gene (SNHG) 7 facilitates NB progression via the microRNA (miR)-653-5p/signal transducer and activator of transcription 2 pathway, providing a novel therapeutic target and prognostic biomarker for NB. The lncRNA family with sequence similarity 201A may affect the radiosensitivity of esophageal squamous cell cancer by regulating ataxia telangiectasia mutated (ATM) and mTOR expression via miR-101 (43). In the present study, RNA-seq demonstrated that 32 up- and 35 downregulated lncRNAs were differentially expressed in the shEZH2 group compared with the control group. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) (44), H19 (45) and X-inactive specific transcript (XIST) (46) were some of the first reported lncRNAs associated with NB development. Koshimizu et al (47) demonstrated that expression level of the tumor marker MALAT1 is sensitive to cell surface receptor activation by oxytocin in an NB cell line. In addition, a six-center case-control study identified three single nucleotide polymorphisms (SNPs; rs2839698 G>A, rs3024270 C>G and rs217727 G>A) from the H19 gene in a Chinese population (700 people with NB and 1,516 controls) and investigated the effect of individual and combined SNPs on NB risk (48). Zhang et al (49) demonstrated that XIST downregulates the Dickkopf Wnt signaling pathway inhibitor 1 by promoting H3 histone methylation via EZH2, inhibiting proliferation, migration and invasion of NB cells and limiting tumor development. In addition, SNHG family members, SNHG5, is upregulated while SNHG15 and SNHG16 is downregulated in NB. SNHG16 is reported to facilitate proliferation, migration, invasion and autophagy of NB cells via sponging miR-542-3p and upregulating autophagy-related 5 expression levels (50). However, the involvement of these lncRNAs in NB remains unknown. Among the 138 lincRNAs identified by EZH2 ChIP-seq, cancer susceptibility 15 was identified as a tumor suppressor that can regulate numerous genes involved in neural crest development (51). GO analysis demonstrated that EZH2 participated in a number of biological processes, such as ‘nervous system development’, ‘regulation of developmental growth’ and ‘histone glutamine methylation’. KEGG analysis showed that ‘Hedgehog signaling pathway’ was enriched in both RIP-seq and RNA-seq, indicating that the pathway may be important in EZH2-associated lincRNAs.

In conclusion, the present study demonstrated that numerous lincRNAs could directly bind to EZH2. Certain lincRNAs may regulate or be regulated by EZH2. Certain lncRNAs were associated with N6-methyladenosine and may potentially encode functional polypeptides. In addition, the difficulty of EZH2-targeted drug research may be associated with these lincRNAs. These lincRNAs may provide a novel option for EZH2-centered molecular target therapy.


Not applicable.


This study received financial support from Shanghai Key Disciplines (grant no. 2017ZZ02022), National Natural Science Foundation of China (grant nos. 81771633 and 81572324) and Science Foundation of Shanghai (grant nos. 17411960600 and 15ZR1404200).

Availability of data and materials

The datasets used during the present study are available from the corresponding author upon reasonable request.

Authors' contributions

KD, DM and MY designed the study. MY, LX and JZ collected the data and performed experiments. BL, XL and JH analyzed and interpreted the data. DM and KD were involved in critical reviewing of the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.





polycomb repressor complex 2


RNA sequencing


RNA immunoprecipitation sequencing


chromatin immunoprecipitation sequencing


Gene Ontology


Kyoto Encyclopedia of Genes and Genomes



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Ye M, Xie L, Zhang J, Liu B, Liu X, He J, Ma D and Dong K: Determination of long non‑coding RNAs associated with EZH2 in neuroblastoma by RIP‑seq, RNA‑seq and ChIP‑seq. Oncol Lett 20: 1, 2020
Ye, M., Xie, L., Zhang, J., Liu, B., Liu, X., He, J. ... Dong, K. (2020). Determination of long non‑coding RNAs associated with EZH2 in neuroblastoma by RIP‑seq, RNA‑seq and ChIP‑seq. Oncology Letters, 20, 1.
Ye, M., Xie, L., Zhang, J., Liu, B., Liu, X., He, J., Ma, D., Dong, K."Determination of long non‑coding RNAs associated with EZH2 in neuroblastoma by RIP‑seq, RNA‑seq and ChIP‑seq". Oncology Letters 20.4 (2020): 1.
Ye, M., Xie, L., Zhang, J., Liu, B., Liu, X., He, J., Ma, D., Dong, K."Determination of long non‑coding RNAs associated with EZH2 in neuroblastoma by RIP‑seq, RNA‑seq and ChIP‑seq". Oncology Letters 20, no. 4 (2020): 1.