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- Ó2019 American Association for Cancer Research. al mRNA processing, assistance of protein localization or www.aacrjournals.org 1305 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst February 4, 2019; DOI: 10.1158/0008-5472.CAN-18-1659 Xu et al. 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 À80C. 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 Â 105 to 8 Â 105 Hep3B