Long non-coding RNA TUG1 is up-regulated in hepatocellular carcinoma and promotes cell growth and apoptosis by epigenetically silencing of KLF2
- Ming-De Huang†1,
- Wen-Ming Chen†2,
- Fu-Zhen Qi†3,
- Ming Sun4,
- Tong-Peng Xu5,
- Pei Ma5 and
- Yong-qian Shu5Email author
© Huang et al. 2015
Received: 11 March 2015
Accepted: 11 August 2015
Published: 4 September 2015
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide, and the biology of this cancer remains poorly understood. Recent evidence indicates that long non-coding RNAs (lncRNAs) are found to be dysregulated in a variety of cancers, including HCC. Taurine Up-regulated Gene 1 (TUG1), a 7.1-kb lncRNA, recruiting and binding to polycomb repressive complex 2 (PRC2), is found to be disregulated in non-small cell lung carcinoma (NSCLC) and esophageal squamous cell carcinoma (ESCC). However, its clinical significance and potential role in HCC remain unclear.
Methods and results
In this study, expression of TUG1 was analyzed in 77 HCC tissues and matched normal tissues by using quantitative polymerase chain reaction (qPCR). TUG1 expression was up-regulated in HCC tissues and the higher expression of TUG1 was significantly correlated with tumor size and Barcelona Clinic Liver Cancer (BCLC) stage. Moreover, silencing of TUG1 expression inhibited HCC cell proliferation, colony formation, tumorigenicity and induced apoptosis in HCC cell lines. We also found that TUG1 overexpression was induced by nuclear transcription factor SP1 and TUG1 could epigeneticly repress Kruppel-like factor 2 (KLF2) transcription in HCC cells by binding with PRC2 and recruiting it to KLF2 promoter region.
Our results suggest that lncRNA TUG1, as a growth regulator, may serve as a new diagnostic biomarker and therapy target for HCC.
Hepatocellular carcinoma (HCC) is the dominant histological type of primary liver cancer which accounts for 70–85 % of primary malignancies in liver, and HCC is the third leading cause of cancer-related death worldwide . While, half of these cases and deaths were estimated to occur in China . Generally, hepatocarcinogenesis is a multistep process involving a number of genetic or epigenetic alterations that eventually result in the malignant transformation of hepatocytes [3, 4]. There have been significant advances in diagnosis and management of HCC and lots of therapeutic strategies have been improved . However, the 5-year overall survival rate remains very poor and the biology of HCC remains poorly understood. Therefore, the identification of the new biomarkers for HCC and a detailed understanding of the molecular mechanisms underlying hepatic carcinogenesis will supply an arm for improving diagnosis and management of human HCC.
MicroRNAs and long noncoding RNAs (lncRNAs) are two major members of ncRNA family, and lots of studies have demonstrated that miRNAs play critical roles in HCC development . For example, miR-331-3p could promote HCC cells proliferation and EMT-mediated metastasis by suppressiing PHLPP-mediated dephosphorylation of AKT . LncRNAs, which are defined as being longer than 200 nucleotides without or with limit protein coding ability [8–10], emerge as essential regulators in almost all aspects of biology via regulation at chromatin organization, transcriptional and post-transcriptional levels [11, 12]. Additionally, a number of studies demonstrate that lncRNAs play an important role in tumorigenesis, and their misexpression confers tumor initiation, cancer cells growth and metastasis [13–15]. For example, lncRNA GAPLINC regulates CD44-dependent cell invasiveness by acting as a molecular decoy for miR211-3p and associates with poor prognosis in gastric cancer . Moreover, lncRNA-ATB activated by TGF-β could promote the invasion -metastasis cascade in HCC cells by binding IL-11 mRNA, autocrine induction of IL-11 and triggering STAT3 signaling . Although, there has been a heavy focus on the ways that lncRNAs contribute to cancers development, but their aberrant expression and functional roles in HCC development and diagnosis are still not well documented.
LncRNA TUG1,a 7.1-kb lncRNA, was firstly detected in a genomic screen for genes up-regulated in response to taurine treatment of developing mouse retinal cells . Recently, TUG1 was found to be generally downregulated in NSCLC . On the contrary,some studies showed that TUG1 can promote the cell proliferation of ESCC , urothelial carcinoma of the bladder  and osteosarcoma . However, the functional role and underlying mechanism of TUG1 in HCC remains unclear. Here we investigated the relationship between TUG1 and HCC. We found that TUG1 was up-regulated in HCC tissues than that in corresponding non-tumor tissues and was related to tumor size and BCLC stage. Moreover, we found that TUG1 overexpression was induced by nuclear transcription factor SP1 and TUG1 could regulate cell growth both in vitro and in vivo via epigenetically silencing KLF2 by binding to PRC2. Our results suggest that TUG1 overexpression was induced by nuclear transcription factor SP1 and TUG1 can regulate KLF2 expression in the epigenetic level and facilitate the development of lncRNA-directed diagnostics and therapeutics of HCC.
TUG1 is up-regulated in hepatocellular carcinoma tissues and is associated with tumor size and BCLC stage
Correlation between TUG1 expression and clinicopathological characteristics of HCC patients
High cases (No 47)
Low cases (No 30)
TUG1 is up-regulated in HCC cell lines and could be activiated by transcript factor SP1
Putative SP1-binding sites in the TUG1 promoter by JASPAR
Predicted site sequence
Knockdown of TUG1 inhibits HCC cell proliferation and induces cell apoptosis in vitro
TUG1 promotes HCC cell proliferation in vivo
TUG1 negatively regulates expression of KLF2
Over-expression of KLF2 impaires HCC cells proliferation and induces cell apoptosis
In the past decade, the discovery of numerous lncRNAs has dramatically altered our understanding of the biology of complex diseases including cancers. Recently, lots of studies have revealed that dysregulated expression of lncRNAs in multiple cancers, which may pinpoint the spectrum of cancer progression and predict patients’ outcome [23, 24]. For example, FAL1 has been identified as an oncogenic lncRNA that associates with BMI1 and represses p21 expression in cancer by a functional genomic approach . In HCC, HULC was the first reported lncRNA that is specifically up-regulated . A number of lncRNAs, such as HULC  and LINC00974  have been reported to be involved in HCC development and progression. In this study, we found that lncRNA TUG1 whose expression is significantly up-regulated in HCC tissues compared with normal tissues. Moreover, increased TUG1 expression was correlated with HCC tumor size and BCLC stage, which suggests that TUG1 may play a key role in HCC development and progression.
Several recent studies indicated that lncRNA expression could also be regulated by some transcript factors (TF), such as lincRNA-p21 expression can be regulated by p53  and TINCR by SP1 . TUG1 expression has been reported to be regulated by an key p53 ; however, we found that TUG1 expression could also be regulated by another TF SP1 in HCC cells, which suggests that one lncRNA may be simultaneously regulated by multiple different transcript factors. As is known, lncRNAs involved in cancer cells biological function, and we found that knockdown of TUG1 could impair HCC cells proliferation, invasion and induce cell apoptosis both in vitro and vivo. These data suggests that lncRNA TUG1 contributes to HCC development via regulation of cell proliferation and apoptosis.
TUG1 has been reported to regulate the expression of HOXB7 in NSCLC . However, we found that TUG1 could bind with both EZH2 and SUZ12 in HCC cells. Furthermore, co-expression analysis indicated that KLF2 could be a new TUG1 downstream target, and knockdown of TUG1, EZH2 and SUZ12 expression indeed both up-regulated KLF2 expression levels in HCC cells. In addition, ChIP assays also demonstrated that EZH2 could directly bind to KLF2 promoter region and inhibition of TUG1 decreased its binding ability. Our results indicated that TUG1 could repress KLF2 transcription by binding with EZH2 and SUZ12 and recruitment of PRC2 to the KLF2 gene locus in HCC cells.
The Kruppel-like factor (KLF) family transcription factors have been identified as suppressors or activators of different genes in a cell type and promoter-dependent manner [31, 32]. Recently, lines of evidence showed that KLF members are emerging as tumor suppressors due to their roles in the inhibition of proliferation, invasion and induction of apoptosis . As an member of KLF family, KLF2 expression is inactivated or lost in several cancers and possesses tumor-suppressor features mediated by KRAS . Moreover, there is evidence showed that EZH2 could directly bind to KLF2 promoter and silence of KLF2 expression result in blocking the tumor-suppressor features of KLF2, which is partly mediated by p21 . Our data also showed that TUG1 could take part in HCC cells proliferation by silencing KLF2 transcription, and KLF2 over-expression further led to the decreased HCC cells proliferation and increased cell apoptosis. Our results suggested that lncRNA, especially TUG1, may influence the same cell biological function via regulating different target genes depending on different cancer cells.
To date, the possible targets and mechanism that underlie lncRNAs mediated regulatory behaviors still remain to be fully investigated in different cancers. In summary, the expression of TUG1 was significantly up-regulated in HCC tissues and cells, suggesting that its overexpression may be an important factor for HCC progression. We showed that TUG1 may regulates the proliferation ability of HCC cells partially through sliencing of the KLF2 by binding with PRC2, which suggested that lncRNAs contribute to different cancer cells biological function through regulating different genes. Our findings further the understanding of HCC pathogenesis, and facilitate the development of lncRNA-directed diagnostics and therapeutics against this disease.
Materials and methods
Patient data and tissue samples
A total of 77 fresh HCC tissue samples and matched normal adjacent tissue samples were selected from patients who underwent resection of primary HCC at Huai’an First People’s Hospital, Nanjing Medical University (Huai’an, China). The HCC diagnosis was histopathologically confirmed. None of the patients received preoperative therapy. Data from all subjects were obtained from medical records, pathology reports, and personal interviews with the subjects. The collected data included age, gender, drinking state, the history of HBV and cirrosis and HCC features (e.g., tumor size, stage). HCC clinical stage was determined according to the BCLC staging classification based on the article by Bruix and Llovet . The clinical information for all of the samples is detailed in Table 1. Fresh samples were snap-frozen in liquid nitrogen immediately after resection and stored at −80 °C. Matched nontumor specimens were obtained from a part of the resected specimen that was farthest from the cancer.
Ethical approval of the study protocol
This study was conducted according to the principles expressed in the Declaration of Helsinki. Tissue specimen collections were made with full informed consent of the patients, and followed institutional ethical guidelines that were reviewed and approved by Huai’an First People’s Hospital, Nanjing Medical University (Huai’an, China).
Human HCC cell lines (HepG2, MHCC-97H, Hep3B) and one normal hepatic epithelial cell line (L02, control) were provided by Dr Beicheng Sun from the Department of Hepatopancreatobiliary, First Affiliated Hospital, Nanjing Medical University (Nanjing City, Jiangsu Province, P. R. China). All cell lines were cultured in DMEM (GIBCO-BRL) medium supplemented with 10 % fetal bovine serum (FBS) at 37 °C in 5 % CO2.
RNA extraction and qPCR analysis
The total RNA was extracted from tissues or cultured cells with TRIzol reagent (Invitrogen, Grand Island, NY, USA), according to the manufacturer’s protocol. One microgram total RNA was reverse transcribed in a final volume of 20 μL under standard conditions using PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Dalian, China; RR047A). After the RT reaction, 1 μL of the complementary DNA was used for subsequent qPCR reactions (SYBR Premix Ex Taq, TaKaRa) following the manufacturer’s protocol. The results were normalized to the expression of GAPDH. The qPCR and data collection were carried out on ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA), and results were analyzed and expressed relative to threshold cycle(CT) values, and then converted to fold changes. All primer sequences are summarized in Additional file 1: Table S1.
Transfection of cell lines
HCC cell lines were transfected with specific siRNA oligonucleotidesby using Lipofectamine RNAi MAX, according to the manufacturer’s protocol (Invitrogen). TUG1 siRNA, to avoid off-target effects and ensure the efficiency of interference, we used two indeed effective interference target sequence of TUG1, according to previous study . EZH2 siRNA was purchased from Realgene (Nanjing, China). Non-specific siRNA (si-NC) was purchased from Invitrogen. Typically, cells were seeded at six-well plates and then transfected the next day with specific siRNA (100 nM) and control siRNA (100 nM). EGFP-SP1 was purchased from Add gene. Plasmid vectors (EGFP-SP1, sh-TUG1 pCMV-Tag2B-FLAG-KLF2 and empty vector) for transfection were prepared using DNA Midiprep or Midiprep kits (Qiagen, Hilden, Germany), and transfected into HepG2 and Hep3B cells.
Cell proliferation assays
Cell proliferation was monitored by Cell Proliferation Reagent Kit I (MTT) (Roche, Basel, Switzerland). The transfected cells were plated in 96-well plates (3000 cells/well). Cell proliferation was determined every 24 h following the manufacturer’s protocol. For the colony-formation assay, 500 transfected cells were placed into each well of a six-well plate and maintained in DMEM containing 12 % FBS for 12 days, replacing the medium every 4 days. Colonies were fixed with methanol and stained with 0.1 % crystal violet (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 15 min. The colony formation was determined by counting the number of stained colonies. Triplicate wells were measured in each treatment group.
Flow cytometry for cell cycle analysis
HepG2 or Hep3B cells for cell cycle analysis were collected 24 h after transfected with si-TUG1 or respective control, 48 h after transfected with pCMV-Tag2B-KLF2 or empty vector. Then cells were stained with PI using the CycleTEST™ PLUS DNA Reagent Kit (BD Biosciences) according to the protocol and analyzed by FACScan. The percentage of the cells in G0/G1, S, and G2/M phase were counted and compared.
Flow cytometry for cell apoptosis analysis
HepG2 or Hep3B cells transfected with si-TUG1, pCMV-Tag2B-KLF2 or respective control were harvested 48 h and then collected. After the double staining with FITC-Annexin V and Propidium iodide (PI) was done using the FITC Annexin V Apoptosis Detection Kit (BD, Biosciences) according to the manufacturer’s protocol, the cells were analyzed with a flow cytometry (FACScan®; BD Biosciences) equipped with a CellQuest software (BD Biosciences). Cells were discriminated into viable cells, dead cells, early apoptotic cells, and apoptotic cells, and then the relative ratio of early apoptotic cells were compared to control transfectant from each experiment.
Cell migration and invasion assays
HepG2 or Hep3B cells transfected with si-TUG1or respective control were harvested 48 h and then collected. For the migration assays, 5 × 104 cells in serum-free medium were placed into the upper chamber of an insert (8 μm pore size; Millipore). For the invasion assays, 1 × 105 cells in serum-free medium were placed into the upper chamber of an insert coated with Matrigel (Sigma-Aldrich). Medium containing 10 % FBS was added to the lower chamber. After incubation for 24 h, we removed the cells remaining on the upper membrane with cotton wool. Cells that had migrated or invaded through the membrane were stained with methanol and 0.1 % crystal violet, imaged, and counted using an IX71 inverted microscope (Olympus, Tokyo, Japan). Experiments were repeated three times.
HepG2 cells were transfected with sh-TUG1 or Scramble using Lipofectamine 2000 (Invitrogen). After 48 h, cells were collected and injected into either side of the posterior flank of the male BALB/c nude mice (4–5 weeks old). Mice were purchased from Shanghai Experimental Animal Center of the Chinese Academy of Sciences. The tumor volumes and weights were measured every 4 days in mice from the control (5 mice) or sh-TUG1 (5 mice) groups, and tumor volumes were calculated by using the equation V = 0.5 × D × d2 (V, volume; D, longitudinal diameter; d, latitudinal diameter). Sixteen days after injection, the mice were killed and tumor weights were measured and used for further analysis. This study was carried out strictly in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Nanjing Medical University.
Tumors from mice were immunostained for HE, ki-67 and KLF2. The signal was amplified and visualized with 3′-diaminobenzidine chromogen, followed by counterstaining with hematoxylin. Expression was considered to be positive when 50 % or more tumor cells were stained. Anti-ki-67(1:50) and anti-KLF2(1:50) were purchased from R&D company.
Western blot assay
The cells were lysed by using mammalian protein extraction reagent RIPA (Beyotime, Haimen, China) supplemented with protease inhibitors cocktail (Roche). Fifty micrograms of the protein extractions were separated by 10 % SDS-PAGE transferred to 0.22 mm nitrocellulose (NC) membranes (Sigma-Aldrich) and incubated with specific antibodies. The autoradiograms were quantified by densitometry (Quantity One software; Bio-Rad, Hercules, CA, USA). Anti-KLF2 was purchased from Sigma (1:1000). Results were normalized to the expression β-actin (Mouse anti-β-actin) (Sigma (1:1000)).
Subcellular fractionation location
The separation of the nuclear and cytosolic fractions of HCC cell lines was performed according to the protocol of the PARIS Kit (Life Technologies, Carlsbad, CA, USA).
Chromatin immunoprecipitation assays(ChIP)
The ChIP assays were performed by using EZ-ChIP KIT according to the manufacturer’s instruction (Millipore, Billerica, MA, USA). HepG2 or Hep3B cells were treated with formaldehyde and incubated for 10 min to generate DNA-protein cross-links. Cell lysates were then sonicated to generate chromatin fragments of 200–300 bp and immunoprecipitated with EZH2 and H3K27me3-specific antibody (CST) or IgG as control. Precipitated chromatin DNA was recovered and analyzed by qPCR.
RIP experiments were performed by using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer’s instructions. Antibody for RIP assays of EZH2 and SUZ12 were purchased from Millipore.
All statistical analyses were performed by using SPSS 17.0 software (IBM, Chicago, IL, USA). The significance of differences between groups was estimated by the Student t-test, Wilcoxon test or χ2 test. Two-sided p-values were calculated, and differences were considered to be statistically significant at P < 0.05. Kendall’s Tau-b and Pearson correlation analyses were used to investigate the correlation between TUG1 and KLF2 expressions.
This study was supported by the National Natural Science Foundation of China (81172140, 81272532), Jiangsu Province Clinical Science and Technology projects (Clinical Research Center, BL2012008) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (Public Health and Preventive Medicine,JX10231801). We are very grateful to Dr Beicheng Sun for providing the HCC cell lines and L02 cell line.
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