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Published OnlineFirst February 4, 2019; DOI: 10.1158/0008-5472.CAN-18-1659

Cancer Genome and Epigenome Research

Genome-Wide Screening and Functional Analysis Identiﬁes Tumor Suppressor Long Noncoding RNAs Epigenetically Silenced in Hepatocellular Carcinoma Feiyue Xu1, Chi Han Li1, Chi Hin Wong1, George G. Chen2, Paul Bo San Lai2, Shengwen Shao3, Stephen L. Chan4, and Yangchao Chen1,5

Abstract

Long noncoding RNAs (lncRNA) play critical roles in the MAPK, and HIF1a pathways that promoted cell proliferation development of cancer, including hepatocellular carcinoma in HCC. Overexpression of EZH2 was critical in repressing (HCC). However, the mechanisms underlying their deregula- TCAM1P-004 and RP11-598D14.1, and EZH2-TCAM1P-004/ tion remain largely unexplored. In this study, we report that RP11-598D14.1–regulated pathways were prevalent in two lncRNAs frequently downregulated in HCC function as human HCC. Aberrant suppression of TCAM1P-004 and tumor suppressors and are epigenetically silenced by histone RP11-598D14.1 led to loss of their tumor-suppressive effects methyltransferase EZH2. lncRNAs TCAM1P-004 and RP11- by disrupting the interaction with IGF2BP1, HIST1H1C, and 598D14.1 were inhibited by EZH-mediated trimethylation STAU1, which in turn promoted HCC development and of H3K27me3 at their promoters. Downregulation of progression. Collectively, these ﬁndings demonstrate the role TCAM1P-004 and RP11-598D14.1 was frequently observed of TCAMP1P-004 and RP11-598D14.1 in suppressing tumor in HCC tumors compared with adjacent normal tissues. Both growth and suggest that EZH2 may serve as a therapeutic target lncRNAs inhibited cell growth, cell survival, and transforma- in HCC. tion in HCC cells in vitro as well as tumor formation in vivo. Using RNA pull-down and mass spectrometry, we demon- Signiﬁcance: EZH2-mediated loss of lncRNAs TCAM1P- strated that TCAM1P-004 bound IGF2BP1 and HIST1H1C, 004 and RP11-598D14.1 hinders the formation of tumor whereas RP11-598D14.1 bound IGF2BP1 and STAU1. These suppressor lncRNA–protein complexes and subsequently lncRNA–protein interactions were critical in regulating p53, promotes HCC growth.

Introduction Africa (5) and more than 50% of diagnosed patients with HCC are Chinese (6). Liver cancer has one of the lowest cancer survival rates (cancer EZH2, a core component of polycomb repressive complex 2 statistics 2018; ref. 1), and more than 80% of liver cancer inci- (PRC2), silences gene expression via its histone methyltransferase dences are hepatocellular carcinomas (HCC; ref. 2). According to activity (7). EZH2 plays essential roles in cancer initiation, progres- the World Health Organization, liver cancer is the second leading sion, and metastasis (8–10). Its expression is elevated aberrantly in cause of death worldwide in 2015 (3). The prognosis of HCC is certain types of cancers including HCC. EZH2 frequently functions very poor with approximately 5%–6% 5-year survival rate (4). as an oncogenic factor and is heavily involved in the silencing of Almost 80% of HCC cases occurred in East Asia and subSaharan critical tumor-suppressive genes such as HOX, CCN3/NOV, DAB2IP, TIMP2/3, CDKN2A/p16, KLF2,andRUNX3 (11, 12). Also, EZH2 participated in the deregulations of miRNAs including miR- 218, miR-26a, miR-1246, and miR-4448 (13, 14). 1School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong. 2Department of Surgery, Prince of Wales Long noncoding RNAs (lncRNA) are noncoding RNA mole- Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong. 3Institute cules with over 200 nucleotides, which are also transcribed by of Microbiology and Immunology, Huzhou University, Huzhou, Zhejiang, China. RNA polymerase II similar to mRNAs (15). LncRNAs play diverse 4Department of Clinical Oncology, State Key Laboratory in Oncology of South roles in biological processes such as epigenetic regulation, trans- China and Institute of Digestive Disease, Prince of Wales Hospital, The Chinese lational regulation, posttranscriptional processing, imprinting, University of Hong Kong, Shatin, Hong Kong. 5Shenzhen Research Institute, The apoptosis, and cell cycle (15, 16). Several aberrantly expressed Chinese University of Hong Kong, Shenzhen, China. lncRNAs have been studied, but only a few lncRNAs have been Note: Supplementary data for this article are available at Cancer Research characterized comprehensively, including HOTAIR, HOTTIP, Online (http://cancerres.aacrjournals.org/). HULC, and MALAT1 (17–20). These lncRNAs have crucial regu- Corresponding Author: Yangchao Chen, The Chinese University of Hong Kong, latory roles in cancer biology. In HCC, aberrant expression of Shatin, Hong Kong. Phone: 852-3943-1100; Fax: 852-2603-5123; E-mail: lncRNAs is frequently observed (21, 22) that results in the [email protected] deregulation of gene expressions through remodeling of chroma- doi: 10.1158/0008-5472.CAN-18-1659 tin, regulation of gene transcription, control of posttranscription- 2019 American Association for Cancer Research. al mRNA processing, assistance of protein localization or

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function, and action in intercellular signaling. The importance of instead of H1 promoter for lncRNA expressions, and coexpressed lncRNAs in cancer is clearly understood, however, the underlying G418-resistant gene and EGFP (10). mechanism of their deregulations is still largely unexplored. Both lncRNAs and EZH2 are important epigenetic regulators in Chromatin immunoprecipitation assay cancer. Numerous protein-coding genes repressed by EZH2 were Chromatin immunoprecipitation (ChIP) assay was performed reported, and the interactions between lncRNAs and EZH2 have by using SimpleChIP Enzymatic Chromatin IP Kit Magnetic Beads been widely studied, but there are few reports studying the (Cell Signaling Technology) according to the manufacturer's regulation of lncRNAs by EZH2. Therefore, we attempted to protocol (23). For immunoprecipitation, anti-EZH2 (Cell Signal- identify the lncRNAs directly regulated by EZH2, reveal the ing Technology; #5246), anti-H3K27me3 (Millipore; 07-449), regulating mechanisms, and explore the roles of these lncRNAs anti-EED (Millipore; 05-1320), anti-SUZ12 (Millipore; 05-1317) in HCC. antibody, and negative control Normal Rabbit IgG antibody were incubated with cross-linked chromatin at 4C overnight with rotation. The precipitated DNA enrichment was quantified by Materials and Methods qPCR and normalized by respective 2% input. Clinical samples, cell lines, and drug treatment Fifty pairs of HCC tumor tissues and adjacent normal tissues siRNA transfection were obtained from patients with HCC who received surgery of Transfections of siRNAs were conducted using DharmaFECT 1 liver cancer resection at the Prince of Wales Hospital, Hong Kong. Transfection Reagent (Thermo Fisher Scientific) according to the The study was carried out according to the ethical guidelines and manufacturer's protocol. During transfection, 100 nmol/L of with the approval of the Joint CUHK-NTEC Clinical Research siRNAs was used to treat the cells for 72 hours before RNA or Ethics Committee in accordance with Declaration of Helsinki and protein extraction. the written informed consent was obtained from all patients recruited. All specimens of HCC tumors and normal tissues were RNA pull-down assay and LC/MS stored at 80C. Each sample was divided into two parts. One was RNA pull-down assay was performed using Pierce Magnetic used for RNA extraction, and the other was fixed and embedded RNA-Protein Pull-Down Kit (Thermo Fisher Scientific) according into paraffin. The nontumorigenic human hepatocyte cell line to the manufacturer's protocol. RNAs were labeled with Biotin MIHA was obtained from Dr. J.R. Chowdhury's laboratory at and were purified by streptavidin magnetic beads. MIHA cell Albert Einstein College of Medicine (New York, NY). The human protein lysates were prepared by using Pierce IP Lysis Buffer and HCC cell line Huh7 (kindly provided by Dr. H. Nakabayashi, incubated with mixture of RNAs and magnetic beads. Unbound Hokkaido University School of Medicine, Sapporo, Japan), Bel- proteins were removed by washing. RNA-binding proteins were 7404, and L02 (Cell Bank of the Chinese Academy of Sciences) purified with acetone and then incubated with sequencing grade were authenticated with short tandem repeat profiling by the trypsin (Promega) to digest protein into peptides at 37C over- vendors. The human HCC cell lines HepG2, PLC/PRF/5 (PLC), night. Peptides were purified by ZipTip Pipette Tips (Merck and Hep3B (ATCC) were verified by short tandem repeat profiling Millipore) according to the manufacturer's protocol. The peptides at the GENEWIZ, Inc. within 6 months of use. Cell lines were were dried using DNA Savant SpeedVac DNA110 (Thermo Fisher maintained in DMEM containing 100 mg/mL streptomycin and Scientific) and detected by LC-Triple TOF 5600 (SCIEX). Results 100 unit/mL penicillin with 10% (v/v) FBS at 37C in a humid- were analyzed by ProteinPilot TM software. ified chamber with 5% CO2. All cell lines undergo routine Myco- plasma testing. For drug treatment, 3-Deazaneplanocin A (DZnep) RNA immunoprecipitation assay was dissolved in ethanol (stock solution concentration 1 mg/mL). RNA immunoprecipitation (RIP) was performed with Magna A total of 5 105 to 8 105 Hep3B and Huh7 cells were seeded in RIP RNA-Binding Protein Immunoprecipitation Kit (Merck Milli- a 12-well plate and were treated with 10 mmol/L DZnep for 48 pore) according to the manufacturer's protocol. Cells were lysed hours. Cells treated with ethanol only were used as the control. in RIP Lysis Buffer with protease inhibitor cocktails and RNase inhibitor. The precipitated RNA was extracted with phenol: Packaging of lentivirus and cell transduction chloroform: isoamyl alcohol or by RNeasy Mini Kit (Qiagen). Packaging of lentivirus was performed according to our pro- qRT-PCR was performed to determine the RNA enrichment with tocol (10). In brief, packaging plasmids pMDLg/pRRE, pRSV-REV, the binding proteins. pCMV-VSVG, and transgene lentiviral were transfected into 293T cells using PEI (polyetherimide, 1 mg/mL). Before transduction, HCC cells (1 104) were seeded on 24-well overnight. 293T cell Results supernatant containing lentivirus particles was added to the wells Identification of upregulated lncRNAs in HCC cells treated with with 10 mg/mL polybrene. Cells were transduced for 24 hours EZH2 inhibitor DZNep before replenishing fresh medium with G418 (800 ng/mL) mam- To identify lncRNAs regulated by EZH2, Huh7 cells and Hep3B malian selection. Lentiviral vector carrying MYC-tagged full cells were treated with 10 mmol/L EZH2 inhibitor DZnep. Treat- length EZH2 was prepared previously (14). Short-hairpin RNA ment with DZnep effectively depleted EZH2 proteins, and H3K27 (shRNA) targeting EZH2, TCAM1P-004, and RP11-598D14.1 trimethylation (H3K27me3) levels were notably reduced in both were cloned into H1 promoter containing lentiviral vectors con- Huh7 and Hep3B cells (Fig. 1A). Arraystar Human lncRNA/ structed by our team that harbored G418-resistant genes and mRNA expression profiling was performed to identify lncRNAs EGFP according to our previous publication (10). Full-length that were differentially expressed after DZnep treatment in Hep3B TCAM1P-004 and RP11-598D14.1 were amplified and cloned cells. We focused on the lncRNAs that passed volcano plot filtering into a same lentiviral backbone containing CMV promoter, with statistical significance (P < 0.05) and had fold change >2.0.

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Figure 1. Identification of two upregulated lncRNAs in HCC cells treated with EZH2 inhibitor DZnep. A, Protein levels of EZH2 and H3K27me3 were decreased in Huh7 cells and Hep3B cells treated with 10 mmol/L DZnep. B, qRT-PCR validation of microarray analysis showed that levels of TCAM1P-004 and RP11–598D14.1 were increased in DZnep-treated HCC cells. C, Their expressions were frequently reduced in HCC cells compared with nontumor MIHA and L02 cells. D, The abundance of TCAM1P-004 and RP11–598D14.1 was lower in human HCC tumor compared with nontumor tissues with statistical significance. E, Kaplan–Meier plots revealed the associations of high TCAM1P-004 and RP11-598D14.1 expressions with a longer overall survival for patients with HCC. Significant differences were analyzed using Mantel–Cox test (n ¼ 50). Results are expressed as the mean SEM. #,, P < 0.05; , P < 0.01; , P < 0.001.

Both upregulation and downregulation of lncRNAs were TCAM1P-004 and RP11-598D14.1 expressions were lower in observed in DZnep-treated cells (Supplementary Table S1). As HCC cells (Fig. 1C). The levels of TCAM1P-004 and RP11- we attempted to identify tumor-suppressive lncRNAs repressed by 598D14.1 in 50 pairs of HCC tumors and corresponding adjacent EZH2, we only focused on the lncRNAs that were upregulated normal tissues were measured by qRT-PCR. Their expression after EZH2 was inhibited by DZnep. We first numbered them with levels were reduced significantly in HCC tumor tissues (P < the prefix DZnep-upregulated (DN). Ten lncRNAs that had the 0.001; Fig. 1D). In addition, Kaplan–Meier plots revealed an highest fold increase from the microarray data were measured in association of higher TCAM1P-004 and RP11-598D14.1 Hep3B and Huh7 cells. We showed that most of them were expression with longer overall survival of patients with HCC upregulated after DZnep treatment (Supplementary Fig. S1), (n ¼ 50; Fig. 1E). Among all ten candidate lncRNAs, only suggesting their specificity to DZnep treatment. We then mea- TCAM1P-004 and RP11-598D14.1 were significantly downregu- sured the upregulated lncRNAs in a panel of HCC and nontumor lated in HCC tissues. Therefore, we omitted other lncRNAs cell lines, and human HCC tissues. LncRNAs that were frequently because their importance was highly diminished as no dysregula- downregulated in HCC cells and tissues were selected for subse- tion in human HCC tissues observed. As such, we focused on quent analysis (Supplementary Figs. S2 and S3). TCAM1P-004 and RP11-598D14.1 in this study. TCAM1P-004 and RP11-598D14.1 (DN2 and DN5, respectively) were two of the ten lncRNAs validated to be upregulated in EZH2 suppressed TCAM1P-004 and RP11-598D14.1 expression Huh7 and Hep3B cells treated with DZnep (Fig. 1B). Their by inducing histone 3 lysine 27 trimethylation expressions were measured in a panel of HCC cell lines including Full-length EZH2 with MYC-tag was cloned into an overexpres- Hep3B, Huh7, Bel7404, and PLC, and in nontumor cell lines sion lentiviral vector (14). EZH2 was ectopically overexpressed MIHA and L02. Compared with MIHA and L02 cells, both in MIHA cells by transducing EZH2 overexpressing lentivirus.

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Figure 2. EZH2 and PRC2 complex inhibited TCAM1P-004 and RP11-598D14.1 expressions. A, Overexpression of EZH2 downregulated TCAM1P-004 and RP11-598D14.1 in MIHA cells. B, Inhibition of EZH2 by shRNAs upregulated both lncRNAs in Hep3B cells. C and D, Transient inhibition of EZH2 by siRNAs upregulated both lncRNAs in Hep3B (C) and Huh7 (D) cells. E and F, Disrupting the formation of PRC2 through inhibiting EED and SUZ12 by siRNAs in Hep3B and Huh7 cells led to the upregulation of TCAM1P-004 and RP11-598D14.1 in both Hep3B (E) and Huh7 (F) cells. , P < 0.05; , P < 0.01; , P < 0.001.

Western blot analysis showed that the EZH2 protein level were transfected with EZH2 siRNAs. The transient inhibition of was higher in the MIHA-EZH2 cells than in the vector control EZH2 by siRNAs also resulted in the upregulation of TCAM1P- cells. MYC-tag expression was observed in the MIHA-EZH2 cells 004 and RP11-598D14.1 in both cell lines (Fig. 2C and D). As the only, indicating that full-length EZH2 was overexpressed in gene silencing effect induced by EZH2 was dependent on the MIHA-EZH2 cells (Fig. 2A). TCAM1P-004 and RP11-598D14.1 polycomb repressive complex 2 (PRC2), we inhibited two PRC2 levels were reduced significantly in EZH2-overexpressing MIHA core members, EED and SUZ12, by siRNAs in Hep3B and Huh7 cells. These indicated that EZH2 was able to repress TCAM1P-004 cells to study their effect on TCAM1P-004 and RP11-598D14.1 and RP11-598D14.1 expressions in nontumor hepatocytes. levels. Inhibition of EED and SUZ12 led to the upregulation of Moreover, we constructed Hep3B-shEZH2 cells transduced by TCAM1P-004 and RP11-598D14.1 in both cell lines (Fig. 2E and lentivirus carrying shRNA targeting EZH2 (14). Western blot F). Taken together, our results suggested that TCAM1P-004 and analysis showed that EZH2 protein was effectively reduced in RP11-598D14.1 were the putative targets repressed by EZH2 in EZH2-inhibited Hep3B cells (Fig. 2B). The expressions of HCC cells. TCAM1P-004 and RP11-598D14.1 were significantly increased EZH2 couples with PRC2 partners to the promoters of protein- in EZH2-inhibited cells compared with the control cells (Fig. 2B). coding genes, thus leading to trimethylation of H3K27 that To further confirm that TCAM1P-004 and RP11-598D14.1 were silences target genes. We hypothesized that EZH2 was recruited the putative targets of EZH2, we measured the expression levels of together with PRC2 members, EED and SUZ12, to regulate TCAM1P-004 and RP11-598D14.1 in Hep3B and Huh7 cells that TCAM1P-004 and RP11-598D14.1. To prove our hypothesis,

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Figure 3. EZH2 recruited PRC2 partners and induced H3K27 trimethylation to suppress TCAM1P-004 and RP11-598D14.1 expression. A, Enrichment levels of EZH2, H3K27me3, SUZ12, and EED at the promoter regions of TCAM1P-004 and RP11-598D14.1 were higher in Hep3B cells compared with MIHA cells. B, Inhibition of EZH2 in Hep3B reduced the occupancies of EZH2, H3K27me3, SUZ12, and EED at the two lncRNAs' promoters. C, Overexpression of EZH2 in MIHA cells increased occupancies of EZH2 and PRC2 partners in the promoters of TCAM1P-004 and RP11-598D14.1. D and E, Inhibition of EED or SUZ12 could lead to the reduced enrichments of EZH2 on the promoter of TCAMP1-004 (D) and RP11-598D14.1 (E). Results are expressed as the mean SEM. , P < 0.05; , P < 0.01; , P < 0.001.

ChIP assays were performed in MIHA and Hep3B cells. ChIP ChIP assays were also carried out in EZH2-inhibited cells. assays showed that EZH2, EED, and SUZ12 were enriched in the Depletion of EZH2 led to the reduced occupancies of SUZ12, promoter regions of TCAM1P-004 and RP11-598D14.1 in Hep3B EED, and H3K27me3 in the promoters of TCAM1P-004 and cells, and the levels were higher than in MIHA cells. Epigenetic RP11-598D14 (Fig. 3B). Overexpression of EZH2 induced the marker H3K27me3, which was catalyzed and maintained by downregulation of TCAM1P-004 and RP11-598D14.1 in MIHA- PRC2, was also detected in the promoters of TCAM1P-004 and EZH2 cells. Upon EZH2 overexpression, the occupancies of RP11-598D14.1 in Hep3B, but not in MIHA cells. This indicated SUZ12, EED, and EZH2, together with H3K27me3 levels at the that TCAM1P-004 and RP11-598D14.1 were both repressed by promoters of TCAM1P-004 and RP11-598D14.1 were also ele- PRC2 in HCC cells, whereas TCAM1P-004 and RP11-598D14.1 vated (Fig. 3C). To demonstrate the involvement of PRC2 com- were not deregulated in MIHA cells with low EZH2 level (Fig. 3A). plex during the repression of TCAM1P-004 and RP11-598D14.1,

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we transiently inhibited EED and SUZ12 by transfecting siRNAs 004 and RP11-598D14.1 could suppress the clonogenic and into Hep3B cells, to study the effects on the targeting of EZH2. transformation ability of HCC cells, as revealed by colony ChIP assay showed that inhibition of EED and SUZ12 could formation assay and soft-agar assay, respectively (Supplemen- hinder the binding of EZH2 on the promoters of TCAM1P-004 tary Fig. S7A and S7B). More importantly, overexpressing and RP11-598D14.1 (Fig. 3D and E). TCAM1P-004 and RP11-598D14.1 in Hep3B cells inhibited the To show that our ChIP assays could identify true interaction rate of tumor formation in vivo. We showed that the size of between EZH2 and target promoter, we attempted to detect the tumors derived from TCAM1P-004 and RP11-598D14.1 over- promoter of a gene nearby TCAM1P-004 genomic region that expressing Hep3B cells were significantly smaller than control named SMARCD2. We showed that SMARCD2 was not regu- group (Fig. 4E, bottom left). IHC staining of proliferation mark- lated by EZH2, as both DZnep treatment and EZH2 siRNAs er KI67 showed that the number of KI67-positive cells was transfection in Hep3B cells were unable to alter SMARCD2 reduced in tumors derived from TCAM1P-004- and RP11- expression (Supplementary Fig. S4A). As expected, we failed to 598D14.1–overexpressing Hep3B cells (Fig. 4E, bottom right). detect the presence of SMARCD2 promoter in the anti-EZH2– These studies demonstrated that TCAM1P-004 and RP11- precipitated DNA as expected (Supplementary Fig. S4B). It 598D14.1 played important roles in tumorigenesis. In turn, proved that our ChIP assay was able to measure the genuine terminal dUTP nick-end labeling (TUNEL) assay was performed interaction between EZH2 and its targeted promoter regions. to study the effect of TCAM1P-004 or RP11-598D14.1 over- These ChIP assays confirmed our hypothesis that EZH2 was expression in the subcutaneous tumors derived. TUNEL assay dependent on the PRC complex to catalyze the formation of showed that apoptosis frequently occurred in tumors derived H3K27me3 and inhibit the transcription of TCAM1P-004 and from TCAM1P-004- or RP11-598D14.1–overexpressing Hep3B RP11-598D14.1. cells (Supplementary Fig. S8). Furthermore, MIHA cells expressing shTCAM1P-004, shRP11-598D14.1, and shSCR were TCAM1P-004 and RP11-598D14.1 acted as tumor suppressors injected subcutaneously into nude mice. The formation of To characterize the biological functions of TCAM1P-004 and tumor was observed after inhibition of TCAM1P-004 and RP11-598D14.1 in HCC, TCAM1P-004 and RP11-598D14.1 were RP11-598D14.1, but not in the control MIHA cells (n ¼ 6; inhibited by at least two independent siRNAs in MIHA cells. MTT Supplementary Fig. S9). Collectively, we demonstrated that cell proliferation assay showed that inhibition of TCAM1P-004 TCAM1P-004 and RP11-598D14.1 negatively regulate HCC and RP11-598D14.1 expression promoted MIHA cell prolifera- growth by inhibiting cell proliferation and inducing apoptosis. tion on day 4 (Supplementary Fig. S5A and S5B). We further knocked-down TCAM1P-004 and RP11-598D14.1 in MIHA Microarray analysis revealed genes and signaling pathways and L02 cells by lentivirus transduction of shRNAs transgenes. related to TCAM1P-004/RP11-598D14.1 MTT assays showed that the proliferation rates of MIHA and L02 To investigate the mechanisms of TCAM1P-004 and RP11- cells were increased after knockdown of TCAM1P-004 and 598D14.1, we measured the changes of gene expression in MIHA RP11-598D14.1 (Fig. 4A–D), and suggested that depletion of cells with TCAM1P-004 or RP11-598D14.1 knockdown by gene TCAM1P-004 or RP11-598D14.1 could promote the growth of expression microarrays. The lists of genes differentially expressed nontumor hepatocytes. in MIHA cells with knockdown of TCAM1P-004 and RP11- Annexin V apoptosis assay was performed to measure 598D14.1 were listed (Supplementary Tables S2 and S3; Supple- the number of apoptotic TCAM1P-004–inhibited or RP11- mentary File S1 and S2). Kyoto Encyclopedia of Genes and 598D14.1–inhibited MIHA cells. There was a nearly 4-fold Genomes (KEGG) pathway analysis showed that the genes dif- decrease in the number of postapoptotic cells in TCAM1P- ferentially expressed after TCAM1P-004 knockdown may be 004–inhibited and RP11-598D14.1–inhibited cells compared involved in the p53 and MAPK signaling pathway (Supplemen- with the controls. This indicated that the inhibitions of the tary Fig. S10A). Similarly, RP11-598D14.1 regulated genes that lncRNAs contributed to the repression of apoptosis in MIHA were associated with the p53, HIF1, and MAPK pathways (Sup- cells (Supplementary Fig. S6A). In turn, we would like to study plementary Fig. S10B). As they were both repressed by EZH2, it the role of TCAM1P-004 and RP11-598D14.1 in the colony- was interesting to analyze the pathway commonly inhibited. forming ability of cancer cells. Colony formation assays Overlapping genes between TCAM1P-004 and RP11-598D14.1 showed that inhibition of TCAM1P-004 and RP11-598D14.1 were used for KEGG pathway analysis, and showed that p53 could promote colony formation in MIHA and L02 cells (Sup- signaling pathway was commonly enriched (Supplementary plementary Fig. S6B and S6C). Moreover, soft-agar assay was Fig. S10C), suggesting that EZH2 heavily deregulated p53 path- used to study the anchorage-independent growth ability of the way through the repression of lncRNAs. qRT-PCR validation also transformed cells. Inhibition of TCAM1P-004 or RP11- indicated that p53-associated genes (GADD45A, CDK6, MDM2, 598D14.1 promoted the anchorage-independent colony for- PMAIP1, and THBS1/TSP1) were downregulated, whereas mation in MIHA cells (Supplementary Fig. S6D), indicating CCNG2 expression was increased. GADD45A and TSP1 func- that TCAM1P-004 and RP11-598D14.1 played a role in inhi- tioned to suppress angiogenesis (24, 25); CDK6, PMAIP1, and biting cell transformation. CCNG2 regulated cell proliferation and apoptosis (26–28). For Subsequently, we would like to study the tumorigenic ability MAPK pathways–associated genes, RAP1A, DUSP4, STK3, DDIT3, of TCAM1P-004 and RP11-598D14.1 in vivo.Hep3BandHuh7 and HSPA1A were downregulated, whereas FOS was upregulated cells were transduced by lentivirus carrying TCAM1P-004 or (Supplementary Fig. S11A). Previous studies reported that RP11-598D14.1 transgenes or blank vector control. MTT assay RAP1A, DDIT3, and HSPA1A regulated cell proliferation (29, showed that there were significant growth-inhibiting effects after 30) and DUSP4 and FOS regulated metastasis (31, 32). In overexpression of TCAM1P-004 and RP11-598D14.1 in both RP11-598D14.1–inhibited cells, genes involved in the p53 path- cell lines (Fig. 4A–D). In addition, overexpression of TCAM1P- way (CDK6, GADD45A, PMAIP1, TSP1, and CCNG2) were

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Figure 4. TCAM1P-004 and RP11-598D14.1 functioned to regulate cell proliferation in hepatocytes and HCC cells. Knockdown of TCAM1P-004 (A) or RP11-598D14.1 (B) promoted cell proliferation in MIHA and L02 cells. Growth rates of TCAM1P-004–overexpressed (C) or RP11-598D14.1–overexpressed HCC (D) cells were decreased compared with control cells. E, Tumor growth for Hep3B cells overexpressed with TCAM1P-004 or RP11-598D14.1 was decreased compared with vector control cells. F, Left, the tumors derived from TCAM1P-004- and RP11-598D14.1–overexpressed cells were smaller than control cells macroscopically. Right, IHC staining showed that tumors derived from TCAM1P-004– and RP11-598D14.1–overexpressed Hep3B cells had reduced number of Ki67-positive cells. , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001.

changed. For genes associated with the HIF1 pathway, only the HIST1H1C in cancer is not explored. To identify the functional expression of PDK4 and PFKFB4 (33, 34) was consistent with the roles of the TCAM1P-004-IGF2BP1, we inhibited IGF2BP1 and microarray data (Supplementary Fig. S11B). HIST1H1C in Hep3B cells with siRNAs (Fig. 5C), and measured the levels of TCAM1P-004 potential targets revealed from previ- TCAM1P-004 interacted with IGF2BP1 and HIST1H1C and ous microarray. DDIT3 was upregulated after knockdown of promoted DDIT3 expression IGF2BP1 in Hep3B cells (Fig. 5D), so we focused on the regulatory LncRNAs can function as signals, decoys, guides, and scaffolds functions of IGF2BP1 on DDIT3. It has been reported that for proteins (35). To determine whether TCAM1P-004 and RP11- IGF2BP1 controls translation of IGF2 (38). IGF2 is associated 598D14.1 exerted their biological functions through protein inter- with the MAPK pathway in which DDIT3 is also involved (39). action, RNA pull-down assays followed by LC/MS were performed Western blot analysis showed that inhibition of IGF2BP1 by to identify RNA-binding proteins that interacted with TCAM1P- transfecting siRNAs significantly upregulated DDIT3 protein 004 and RP11-598D14.1. Proteins bound to TCAM1P-004 with levels (Fig. 5E). After revealing the association between IGF2BP1 positive detection and showed greater than 95% confidence inter- and DDIT3, we studied the changes in protein levels of IGF2BP1, val (CI) were listed (Supplementary Table S4). By literature review, IGF2, and DDIT3 in MIHA and L02 cells after TCAM1P-004 was we selected the potential binding proteins that were associated inhibited. Although inhibition of TCAM1P-004 had no effect on with cancer for further validation. Western blot analysis validated IGF2BP1, the IGF2 level was increased while DDIT3 was reduced that IGF2BP1 and HIST1H1C were detected from TCAM1P-004– in both TCAM1P-004–inhibited MIHA and L02 cells (Fig. 5E). In bound proteins but not the negative control (Fig. 5A). RIP assay turn, we measured DDIT3 expression in HCC cell lines and HCC also showed that TCAM1P-004 was obviously enriched with tissues. Compared with MIHA cells, mRNA expression of DDIT3 IGF2BP1 and HIST1H1C compared with IgG control (Fig. 5B). was downregulated in five different HCC cell lines (Supplemen- IGF2BP1 is an RNA-binding protein that functions as a protu- tary Fig. S12A). DDIT3 mRNA levels were also significantly morigenic factor in HCC (36). It was reported that IGF2BP1 could decreased in 50 pairs of HCC tumor compared with adjacent bind with c-MYC and MKI67 mRNAs to enhance their protein normal tissues (Supplementary Fig. S12B). Correlation analysis expressions (36). IGF2BP1 also bound with lncRNA HCG11 to showed that TCAM1P-004 expression was positively associated suppress HCC cell apoptosis (37). Meanwhile, the role of with DDIT3 expression (R2 ¼ 0.1975; P < 0.0001; Supplementary

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Figure 5. TCAM1P-004 interacted with IGF2BP1 and HIST1H1C and activated DDIT3 expression. A, In vitro RNA-protein binding assay and mass spectrometry were conducted to identify TCAM1P-004–interacting proteins. IGF2BP1 and HIST1H1C were validated to be the binding proteins of TCAM1P-004 by Western blot analysis. B, RIP showed that IGF2BP1 and HIST1H1C interacted endogenously with TCAM1P-004. C, Inhibition efﬁciency of siRNAs targeting IGF2BP1 in Hep3B cells were validated by qRT-PCR. D, TCAM1P-004–related genes involved in MAPK pathway were measured in IGF2BP1-inhibited Hep3B cells. Knockdown of IGF2BP1 increased mRNA levels of GADD45A and DDIT3 in Hep3B cells. E, Upon knockdown of IGF2BP1, the DDIT3 protein level was upregulated while IGF2 protein level was downregulated in Hep3B cells. In TCAM1P-004–inhibited MIHA and L02 cells, IGF2 protein level was increased while DDIT3 protein level was reduced, but there was no change in IGF2BP1 expression. F, mRNA and protein levels of DDIT3 were downregulated in HIST1H1C-inhibited Hep3B and Huh7 cells. Results are expressed as the mean SEM. , P < 0.05; , P < 0.01; , P < 0.001.

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Tumor Suppressor lncRNAs Epigenetically Silenced in HCC

Figure 6. RP11-598D14.1 interacted with IGF2BP1 and STAU1. A, In vitro RNA-protein binding assay and mass spectrometry were conducted to identify RP11-598D14.1– interacting proteins. Western blotting validated that IGF2BP1 and STAU1 were the binding proteins of RP11-598D14.1. B, RIP showed that IGF2BP1 and STAU1 interacted endogenously with RP11-598D14.1. C, mRNA level and protein level of DDIT3 were reduced in RP11-598D14.1–inhibited MIHA cells. D, Knockdown of STAU1 increased PFKFB4 mRNA expression in Hep3B cells. E, Knockdown of RP11-598D14.1 increased PFKFB4 protein level. STAU1 protein had no change in RP11-598D14.1–inhibited MIHA cells. Results are expressed as the mean SEM. , P < 0.05; , P < 0.01; , P < 0.001.

Fig. S12C). Collectively, our data suggested that TCAM1P-004 expression, whereas IGF2 protein levels were increased and interacted with IGF2BP1 to repress IGF2 translation and in turn DDIT3 protein levels were reduced upon the depletion of promoted DDIT3 expression. RP11-598D14.1 expression (Fig. 6C). Correlation analysis We next explored the interaction between TCAM1P-004 and showed that RP11-598D14.1 expression was positively associated HIST1H1C. First, we knocked-down HIST1H1C in Hep3B and with DDIT3 expression (R2 ¼ 0.2091 and P < 0.0001; Supple- Huh7 cells with siRNAs. Then, we measured the expression of mentary Fig. S12D). Therefore, we suggested that RP11- TCAM1P-004 potential target genes revealed from previous gene 598D14.1-IGF2BP1 regulated IGF2 expression that led to the expression microarray. We showed that DDIT3 expression was downregulation of DDIT3 expression. again downregulated in Hep3B and Huh7 cells after inhibition of STAU1 is another RNA-binding protein that has an important HIST1H1C. Western blot analysis results conﬁrmed the inhibitory role in cancer. It was reported that STAU1 could bind to lncRNA effects of HIST1H1C on DDIT3 in HCC cells, indicating that TINCR and promoted gastric cancer progression by regulating the interaction between TCAM1P-004 and HIST1H1C also con- KLF2 mRNA stability (40). STAU1 also bound to lncRNA SNHG5 tributed to the repression of DDIT3 (Fig. 5F). to promote colorectal cancer cell survival (41). However, the role of STAU1 in HCC is not studied. To investigate the biological RP11-598D14.1 interacted with IGF2BP1 and STAU1 function of the RP11-598D14.1–STAU1 interaction, we measured Proteins bound to RP11-598D14.1 that were detected by RNA the changes in expression of RP11-598D14.1–targeted genes after pull-down LC/MS were listed (with positive detection and over knockdown of STAU1 in Hep3B. Expression of SLC2A3, EGLN3, 95% CI; Supplementary Table S5). Subsequent Western blot TNSF15, CCNG2, and PFKFB4 were signiﬁcantly upregulated analysis validated that RP11-598D14.1 was able to bind with after inhibition of STAU1 (Fig. 6D). Western blot analysis revealed IGF2BP1 and STAU1 (Fig. 6A). RIP assay showed that the endog- that STAU1 protein level did not change after the knockdown of enous interaction of IGF2BP1 and STAU1 with RP11-598D14.1 RP11-598D14.1 in MIHA cells, whereas PFKFB4 expression was could be observed in MIHA cells (Fig. 6B). Because RP11- upregulated upon the knockdown of RP11-598D14.1 expression 598D14.1 was shown to bind with IGF2BP1, we speculated that (Fig. 6E). This provided evidence that RP11-598D14.1–STAU1 RP11-598D14.1 could also regulate IGF2 translation and DDIT3 complex could repress PFKFB4 expression. PFKFB4 was reported expression. First, we measured the mRNA level of DDIT3 in RP11- to be an oncogene in cancer (42, 43), and The Cancer Genome 598D14.1–inhibited MIHA cells and found that DDIT3 expres- Atlas data analysis showed that PFKFB4 level was higher in HCC sion was reduced compared with that in the control cells (Fig. 6C). tumor tissues compared with nontumor tissues (Supplementary Inhibition of RP11-598D14.1 had no effect on IGF2BP1 protein Fig. S13). It indicated that PFKFB4 should play an important role

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Figure 7. Knockdown of IGF2BP1 and HISIT1HIC suppressed HCC cell growth. A, qRT-PCR showed that TCAM1P-004 and RP11-598D14.1 were upregulated in the TCAM1P- 004–overexpressed tumor and RP11-598D14.1–overexpressed tumor, respectively. B, PFKFB4 was signiﬁcantly downregulated in RP11-598D14.1–overexpressed tumor. C, DDIT3 expressions were signiﬁcantly increased in both TCAM1P-004–overexpressed and RP11-598D14.1–overexpressed tumors. D, MTT assay showed that inhibition of IGF2BP1 could repress cell proliferation in Hep3B and Huh7 cells. E, Cell proliferation of Hep3B cells and Huh7 cells was suppressed after treatment with siRNAs targeting HIST1H1C. Results are expressed as the mean SEM. F, Hypothetical model of tumor-suppressive roles of TCAM1P-004 and RP11-598D14.1 in HCC. EZH2 coupled with PRC2 partners to silence TCAM1P-004 and RP11-598D14.1 through inducing H3K27me3. Downregulation of TCAM1P- 004 and RP11-598D14.1 resulted in the suboptimal level of TCAM1P-004-IGF2BP1, RP11-598D14.1-IGF2BP1, and TCAM1P-004-HIST1H1C, which subsequently decreased DDIT3 expression and promoted PFKFB4 expression. , P < 0.05; , P < 0.01; , P < 0.001.

in HCC and the silencing of RP11-598D14.1 might contribute to 598D14.1 inhibited PFKBF4 level in vivo. Moreover, TCAM1P-004 the upregulation of PFKFB4 in human HCC. and RP11-598D14.1 could couple with IGF2BP1 to reduce the protein level of IGF2, and subsequently increase the expression of IGF2BP1 and HIST1H1C promoted cell proliferation in HCC DDIT3 in vitro. qRT-PCR showed that DDIT3 expressions were To show that the lncRNA-regulatory axis was relevant in vivo,we significantly increased in both TCAM1P-004–overexpressed and measured the expression of downstream genes in Hep3B xeno- RP11-598D14.1–overexpressed tumors (Fig. 7C). These evi- graft tumors. tRNA was extracted from tumors derived from dences suggested that the lncRNA-regulatory pathways identified TCAM1P-004- and RP11-598D14.1-overexpressed Hep3B cells were functioning in vivo. Furthermore, we also investigated the and the vector control cells. qRT-PCR showed that TCAM1P-004 biological functions of IGF2BP1 and HIST1H1C in HCC cells and RP11-598D14.1 were upregulated in the TCAM1P-004–over- through knockdown of their expression by siRNAs. The cell expressing and RP11-598D14.1–overexpressing tumors, respec- proliferation of Hep3B and Huh7 cells were hindered after the tively (Fig. 7A). We had previously shown that RP11-598D14.1 knockdown of IGF2BP1 or HIST1H1C (Fig. 7D and E). Taken could couple with STAU1 to inhibit PFKFB4. qRT-PCR showed together, we demonstrated that IGF2BP1 and HIST1H1C pro- that there was a significant downregulation of PFKFB4 in RP11- moted cell proliferation in HCC and their functions could be 598D14.1–overexpressing tumor (Fig. 7B), suggesting that RP11- regulated by TCAM1P-004 and RP11-598D14.1.

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Discussion molecular functions independent of TCAM1P-004 that promoted cell proliferation. In this study, we revealed a novel function of EZH2 in the Here, we demonstrated that DDIT3 was one of the critical repression of lncRNAs TCAM1P-004 and RP11-598D14.1 in HCC proteins repressed upon the loss of TCAM1P-004 and RP11- and expanded the landscape of the epigenetic regulatory roles of 598D14.1. DDIT3, also called CHOP and GADD153, is a key EZH2 and lncRNAs in HCC. The schematic diagram demonstrat- regulator under cellular stress. Studies showed that DDIT3 reg- ed the mechanism in promoting HCC growth via the aberrant ulates cell migration, proliferation, cell apoptosis and survival in suppression of TCAM1P-004 and RP11-598D14.1 by EZH2 many cancers (47–49). Low DDIT3 expression in cell lines and (Fig. 7F). EZH2 within the PRC2 complex bound to the promoters human samples may imply a critical role of DDIT3 in HCC. of TCAM1P-004 and RP11-598D14.1, resulted in the epigenetic Furthermore, we found that PFKFB4 may be the downstream silencing of both lncRNAs. Downregulation of TCAM1P-004 targeting gene of the RP11-598D14.1–STAU1 complex. PFKFB4 is and RP11-598D14.1 reduced the level of lncRNA–protein reported to be an oncogene in glioblastoma and bladder can- complexes including TCAM1P-004-IGF2BP1, TCAM1P-004- cer (42, 43). The Cancer Genome Atlas analysis also showed that HIST1H1C, RP11-598D14.1-IGF2BP1 and RP11-598D14.1- upregulation of PFKFB4 expression was frequently observed in STAU1. IGF2BP1, HIST1H1C and STAU1 released from the HCC tumor samples. Currently, the role of PFKFB4 in HCC was interactions from TCAM1P-004 and RP11-598D14.1 could then largely unexplored. Further characterization of this protein may repressed proapoptotic factor DDIT3 or promoted antiapoptotic reveal its importance in human HCC. factor PFKFB4 that heavily contributed to HCC tumorigenesis In addition to EZH2, EZH1 was another mammalian homolog (Fig. 7F). in the EZH family that served similar molecular functions, but IGF2BP1 belongs to the conserved protein family IGF2BP showed distinct expression pattern in different developmental (44). The IGF2BP family proteins were heavily involved in stages. There was no study reporting that EZH1 had any role in the cancer biology, and high expression of IGF2BP causes an development or progression of HCC. To exclude the potential aggressive malignancy phenotype (44). Consistently, we also involvement of EZH1 in human HCC, we measured the EZH1 observed that IGF2BP1 could promote cell proliferation in mRNA expression in a panel of HCC cell lines and nontumor HCC cells. As TCAM1P-004 and RP11-598D14.1 participated hepatocyte cell lines, and showed that the level of EZH1 expres- in the suppression of cell proliferation, we hypothesized that sion had no significant difference between HCC and nontumor TCAM1P-004 and RP11-598D14.1 could block the biological cells (Supplementary Fig. S15A). We also attempted to exclude the function of IGF2BP1 through physical interaction. The ability unspecific effect of DZnep on EZH1, and showed that DZnep of TCAM1P-004 and RP11-598D14.1 competing to bind with treatment had no effect on the mRNA level of EZH1 (Supple- oncogenic RNA-binding proteins underlay parts of their tumor- mentary Fig. S15B). Indeed, we had performed alternative suppressive functions. Here, the interactions of TCAM1P-004 approaches to specifically inhibit EZH2 in HCC cells (e.g., shRNAs and RP11-598D14.1 with IGF2BP1 resulted in the disruption of and siRNAs targeting EZH2-specific regions), and demonstrated IGF2BP1-promoted IGF2 translation that led to promotion of that the affected lncRNAs were putative EZH2-regulating targets. cell apoptosis and reduction of cell proliferation. As both Collectively, this work further elucidates the biological roles of TCAM1P-004 and RP11-598D14.1 were able to bind with EZH2. EZH2 not only regulates protein-coding genes and miRNAs IGF2BP1, so they were expected to share certain degree of expression, but also suppresses the expression of lncRNAs, sequence homology. However, alignment of the two lncRNAs TCAM1P-004 and RP11-598D14.1. Because TCAM1P-004 and showed low similarity (29.7%) on their RNA sequences (Sup- RP11-598D14.1 were significantly reduced in human HCC sam- plementary Fig. S14), suggesting that they might not share ples and negatively associated with survival time of patients with similar RNA structure or motif. The IGF2BP1 has two RNA HCC, TCAM1P-004 and RP11-598D14.1 could be potential recognition motifs and a RGG RNA-binding domain, which diagnostic markers for HCC, which could help to identify HCC show different degree of preference for RNA but often display at the early stage. More importantly, TCAM1P-004 and RP11- degenerate binding specificity. Given that IGF2 signaling was 598D14.1 played tumor-suppressive functions by inhibiting cell critical in carcinogenesis, multilevel of regulations should be proliferation and tumor growth. The association of cell apoptosis present to tightly control it in nontumor cells. Thus, it is not with the EZH2-TCAM1P-004/IGF2BP1-IGF2-DDIT3, EZH2- surprising that multiple lncRNAs could function to regulate the RP11-598D14.1/IGF2BP1-IGF2-DDIT3, and EZH2-TCAM1P- IGF2 signaling. 004/HIST1H1C-DDIT3 pathways suggests that TCAM1P-004 and The other TCAM1P-004-binding protein HIST1H1C, which RP11-598D14.1 could be used in the development of a novel belongs to the histone H1 family, plays important roles in various therapeutic strategy. cancers. Song and colleagues observed that HIST1H1C is the downstream targeting gene of HSP 90 in pancreatic cancer (45). Disclosure of Potential Conflicts of Interest In human breast cancer cells, HIST1H1C was found to have low No potential conflicts of interest were disclosed. abundance at the transcription start sites of the inactive genes and was proven to be intensively correlated with low gene expres- Authors' Contributions sion (46). In HCC, we observed that knockdown of HIST1H1C Conception and design: F. Xu, Y. Chen caused a decreased expression of DDIT3, indicating that Development of methodology: F. Xu, P.B.S. Lai HIST1H1C interaction with TCAM1P-004 may promote DDIT3 Acquisition of data (provided animals, acquired and managed patients, expression. Although the TCAM1P-004–HIST1H1C complex provided facilities, etc.): F. Xu, S.L. Chan Analysis and interpretation of data (e.g., statistical analysis, biostatistics, could downregulate DDIT3 expression, which induced cell apo- computational analysis): F. Xu, C.H. Li, P.B.S. Lai, Y. Chen ptosis, inhibition of HIST1H1C suppressed cell proliferation in Writing, review, and/or revision of the manuscript: F. Xu, C.H. Li, P.B.S. Lai, both Hep3B and Huh7 cells. HIST1H1C may exhibit alternative S.L. Chan, Y. Chen

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Administrative, technical, or material support (i.e., reporting or organizing Foundation of China (81427340), and Direct Grant from CUHK data, constructing databases): C.H. Li, C.H. Wong, G.G. Chen, S. Shao, (to Y. Chen). S.L. Chan, Y. Chen Study supervision: Y. Chen 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 Acknowledgments accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The work described in this article was supported by grants from the General Research Fund, Research Grants Council of Hong Kong Received May 31, 2018; revised September 27, 2018; accepted January 31, (CUHK462713, 14102714, and 14136416), National Natural Science 2019; published ﬁrst February 4, 2019.

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Genome-Wide Screening and Functional Analysis Identifies Tumor Suppressor Long Noncoding RNAs Epigenetically Silenced in Hepatocellular Carcinoma

Feiyue Xu, Chi Han Li, Chi Hin Wong, et al.

Cancer Res 2019;79:1305-1317. Published OnlineFirst February 4, 2019.

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