- Email: [email protected]
EBPβ-LINC01133 axis promotes cell proliferation in pancreatic ductal adenocarcinoma through upregulation of CCNG1
Accepted Manuscript The C/EBPβ-LINC01133 axis promotes cell proliferation in pancreatic ductal adenocarcinoma through upregulation of CCNG1 Chen-Song Huang, Junjun Chu, Xiao-Xu Zhu, Jian-Hui Li, Xi-Tai Huang, Jian-Peng Cai, Wei Zhao, Xiao-Yu Yin PII:
S0304-3835(18)30153-8
DOI:
10.1016/j.canlet.2018.02.020
Reference:
CAN 13770
To appear in:
Cancer Letters
Received Date: 8 January 2018 Revised Date:
11 February 2018
Accepted Date: 12 February 2018
Please cite this article as: C.-S. Huang, J. Chu, X.-X. Zhu, J.-H. Li, X.-T. Huang, J.-P. Cai, W. Zhao, X.Y. Yin, The C/EBPβ-LINC01133 axis promotes cell proliferation in pancreatic ductal adenocarcinoma through upregulation of CCNG1, Cancer Letters (2018), doi: 10.1016/j.canlet.2018.02.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Abstract Long non-coding RNAs (lncRNAs) are emerging as important regulators and prognostic markers of multiple cancers. Our aim was to determine functional
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involvement of lncRNAs in pancreatic ductal adenocarcinoma (PDAC). In this study, we report that LINC01133 expression is higher in PDAC tissues compared to adjacent non-cancerous tissues, and this overexpression is associated with poorer prognosis
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among the patients. In vitro, a knockdown of LINC01133 substantially decreased
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PDAC cell proliferation. Tumorigenicity of PDAC cells with the LINC01133 knockdown was significantly impaired in a xenograft model assay. Moreover, we determined that CCAAT/enhancer-binding protein β (C/EBPβ) positively regulates LINC01133 expression by binding to the response elements within the LINC01133
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promoter. Higher expression of C/EBPβ was observed in PDAC tissues, and this overexpression was also associated with the poorer prognosis. Furthermore, the LINC01133 knockdown decreased cyclin G1 (CCNG1) expression. Overexpression
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of CCNG1 attenuated the LINC01133 silencing–induced impairment of proliferation
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in PDAC cells. In summary, our findings revealed that the C/EBPβ-LINC01133 axis performs an oncogenic function in PDAC by activating CCNG1, which may serve as a prognostic biomarker or a therapeutic target in PDAC.
ACCEPTED MANUSCRIPT The C/EBPβ-LINC01133 axis promotes cell proliferation in pancreatic ductal
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adenocarcinoma through upregulation of CCNG1
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Chen-Song Huang1#, Junjun Chu2#, Xiao-Xu Zhu1, Jian-Hui Li1, Xi-Tai Huang1,
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Jian-Peng Cai1, Wei Zhao2*, Xiao-Yu Yin1*
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Yat-sen University, Guangzhou 510080, China
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# These authors contributed equally to this work.
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Department of Pancreato-Biliary Surgery, The First Affiliated Hospital of Sun
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Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China.
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Abbreviations: LncRNAs, Long non-coding RNAs; PDAC, pancreatic ductal
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adenocarcinoma; C/EBPβ, CCAAT/enhancer-binding protein β; CCNG1, cyclin
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protein G1; DFS, disease-free survival; OS, overall survival; siRNA, small interfering
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RNA; qRT-PCR, quantitative real-time PCR; ChIP, chromatin immunoprecipitation;
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GEO, Gene Expression Omnibus; PDAC, pancreatic ductal adenocarcinoma; shRNA,
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short hairpin RNA; TCGA, The Cancer Genome Atlas
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*Correspondence
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Yin XY (email: [email protected])
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Zhao W (email: [email protected])
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ACCEPTED MANUSCRIPT Abstract
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Long non-coding RNAs (lncRNAs) are emerging as important regulators and
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prognostic markers of multiple cancers. Our aim was to determine functional
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involvement of lncRNAs in pancreatic ductal adenocarcinoma (PDAC). In this study,
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we report that LINC01133 expression is higher in PDAC tissues compared to adjacent
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non-cancerous tissues, and this overexpression is associated with poorer prognosis
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among the patients. In vitro, a knockdown of LINC01133 substantially decreased
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PDAC cell proliferation. Tumorigenicity of PDAC cells with the LINC01133
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knockdown was significantly impaired in a xenograft model assay. Moreover, we
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determined that CCAAT/enhancer-binding protein β (C/EBPβ) positively regulates
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LINC01133 expression by binding to the response elements within the LINC01133
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promoter. Higher expression of C/EBPβ was observed in PDAC tissues, and this
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overexpression was also associated with the poorer prognosis. Furthermore, the
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LINC01133 knockdown decreased cyclin G1 (CCNG1) expression. Overexpression
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of CCNG1 attenuated the LINC01133 silencing–induced impairment of proliferation
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in PDAC cells. In summary, our findings revealed that the C/EBPβ-LINC01133 axis
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performs an oncogenic function in PDAC by activating CCNG1, which may serve as
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a prognostic biomarker or a therapeutic target in PDAC.
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Keywords: LINC01133; CCAAT/enhancer-binding protein β; Cyclin G1; Pancreatic
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ductal adenocarcinoma; PDAC prognostic biomarker.
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ACCEPTED MANUSCRIPT 1. Introduction
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Pancreatic ductal adenocarcinoma (PDAC), accounting for more than 90% of
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pancreatic cancer cases, is the fourth leading cause of death due to cancer in the
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United States [1]. Only 15–20% of patients with pancreatic cancer have a diagnosis of
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resectable disease. Patients with locally advanced cancer or metastasis have poor
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prognosis [2,3]. Even though a large number of signaling pathways, growth factors,
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oncogenes, and tumor suppressor genes have been found to participate in the initiation
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and progression of pancreatic cancer [4], few of them are known to be useful for the
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treatment of pancreatic cancer [5]. Therefore, identification of key regulators that
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control PDAC carcinogenesis and progression is critically important for developing
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more effective diagnostics and therapeutics.
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Accumulating evidence reveals that long non-coding RNAs (lncRNAs) play
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important roles in the carcinogenesis and progression of cancer. LncRNA GClnc1
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performs a tumorigenic function by recruiting the WDR5 and KAT2A complex and by
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modifying the transcription of SOD2 in human gastric cancer [6]. LncRNA ATB
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upregulates ZEB1 and ZEB2 by competitively binding to members of the
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microRNA-200 (miR-200) family and then induces epithelial–mesenchymal transition
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and invasiveness of hepatocellular carcinoma [7]. Recently, another study showed that
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LINC00673 reinforces the interaction of PTPN11 with PRPF19 and promotes
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PTPN11 degradation through ubiquitination, which causes diminished SRC-ERK
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oncogenic signaling and enhances activation of the STAT1-dependent antitumor
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response [8]. Although several published studies have shown the involvement of
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ACCEPTED MANUSCRIPT lncRNAs in PDAC, how lncRNAs promote PDAC proliferation remains unclear. In
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addition, the mechanism of lncRNAs’ action on PDAC carcinogenesis needs to be
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further teased out.
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In the present study, we investigated the pathological role of LINC01133 in human
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PDAC. LINC01133 levels were found to be remarkably upregulated in human PDAC
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tumor tissues compared to adjacent non-cancerous tissues; this upregulation positively
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correlated with the poor prognosis and shorter survival time. Both in vivo and in vitro
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experiments revealed that LINC01133 promoted PDAC cell proliferation.
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CCAAT/enhancer-binding protein β (C/EBPβ), a regulator of cell proliferation,
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controls the expression of LINC01133 by directly binding to its promoter.
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LINC01133 next activates the transcription of cyclin protein G1 (CCNG1) and
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induces the proliferation and growth of cancer cells. Thus, our study revealed that the
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C/EBPβ-LINC01133 axis may act as a prognostic biomarker or a novel therapeutic
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target in PDAC.
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ACCEPTED MANUSCRIPT 2. Materials and methods
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2.1. Patients’ specimens
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A total of 132 histologically proved PDAC patients, who underwent surgical resection
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of PDAC at the First Affiliated Hospital of Sun Yat-sen University (Guangzhou,
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China), were recruited into the study. Among them, 83 PDAC patients, who
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underwent pancreatectomy between January 2008 and December 2013, were recruited
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for the clinicopathological and prognostic analysis. The inclusion criteria were as
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follows: (1) undergoing curative resection; (2) receiving no preoperative
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chemotherapy; (3) the absence of distant metastasis; (4) surviving longer than 30 days
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after operation; (5) having the integrated clinicopathological data and follow-up data;
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(6) availability of a tumor tissue specimen. The clinicopathological characteristics are
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shown in Supplementary Table 1. In the remaining 49 patients who underwent
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pancreatectomy between January 2015 and May 2017, tumorous and adjacent
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non-cancerous tissues were obtained, snap-frozen instantly in liquid nitrogen, and
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stored at −80°C until RNA extraction and quantitative real-time PCR for analysis of
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LINC01133 expression. This study’s protocol was approved by the Ethics Committee
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of the First Affiliated Hospital of Sun Yat-sen University. Written informed consent
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was obtained from each patient.
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2.2. Extraction and processing of Gene Expression Omnibus (GEO) and The Cancer
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Genome Atlas (TCGA) data
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Three Affymetrix® Human Genome U133-plus2 microarray datasets (GSE15471, 5
ACCEPTED MANUSCRIPT GSE16515, and GSE32676) were selected, and the raw data were downloaded from
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the GEO database (https://www.ncbi.nlm.nih.gov/gds/). The array data of GSE15471
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[9] consisted of 36 PDAC tissue samples and 36 adjacent non-cancerous tissue
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samples. The array data of GSE16515 [10] included 36 PDAC tissue samples and 16
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adjacent non-cancerous tissue samples. The dataset of GSE32676 [11] included 25
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PDAC tissue samples and 7 adjacent non-cancerous tissue samples. The .cel files of
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each sample were downloaded and analyzed using the “affy” package in R 3.4.1. A
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total of 3989 probes have been selected and annotated as lncRNAs. The expression
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values of all those probes were extracted from the normalized expression data of the
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whole array. Then, lncRNA probes differentially expressed between PDAC tissue and
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adjacent non-cancerous tissue were determined by an empirical Bayes statistics
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method of the “limma” package in R 3.4.1. The representative probe of LINC01133 is
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239370_at.
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TCGA data on LINC01133, C/EBPβ, and CCNG1 expression in PDAC tissues and
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clinical
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(https://tcga-data.nci.nih.gov/tcga/). LINC01133 expression in PDAC and its
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correlations with clinical parameters of patients with PDAC were analyzed. The
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Kaplan-Meier survival curves were constructed to examine the impact of the
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LINC01133 gene on the disease-free survival (DFS) and overall survival (OS) of
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PDAC patients.
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ACCEPTED MANUSCRIPT 2.3. Immunohistochemical (IHC) staining
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PDAC tissues of 83 patients obtained from the Department of Pathology of the First
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Affiliated Hospital of Sun Yat-sen University and PDAC tumors from respective
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groups of xenograft nude mice were paraffin-embedded for IHC staining. This
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staining was performed as described previously [12]. Two experienced pathologists
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independently scored the stained tissues according to both the area of positive staining
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and the intensity of staining. Cutoff values were chosen on the basis of heterogeneity
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measurement by the log-rank test with respect to OS and DFS.
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2.4. Reagents & antibodies
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Small interfering RNAs (siRNAs) targeting human LINC01133, C/EBPβ, or CCNG1
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and non-targeting control siRNA were purchased (Genepharma, Suzhou, China). The
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pReceiver-M98-C/EBPβ overexpression plasmid and empty vector pReceiver-M98
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were purchased from Genecopoeia (Rockville, MD). Human LINC01133 small
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hairpin RNA (shRNA) was ligated into the UUP vector to construct the LINC01133
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shLINC01133 plasmid. Human CCNG1 cDNA was ligated into the pcDNA3.1 vector
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to construct the pcDNA3.1-CCNG1 overexpression plasmid. The following
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antibodies against the indicated proteins were employed in this study: a rabbit
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anti-human C/EBPβ antibody [Abcam, UK], rabbit anti-human Ki-67 antibody
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[ProteinTech Group, USA], and rabbit anti-human CCNG1 antibody [ProteinTech
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Group, USA]. The sequences of primers, probes, shRNA, and siRNA used for the
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experiments in this study are listed in Supplementary Table 2.
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2.5. Cell culture and transfection
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Four PDAC cell lines (BXPC3, CFPAC1, PANC1, and SW1990) were purchased 7
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Academy of Sciences (Shanghai, China). PDAC cell line CAPAN-2 was acquired
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from the American Type Culture Collection (Manassas, VA, USA). BXPC3 cells
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were cultured in RPMI 1640 (Gibco, USA); CFPAC1 cells were cultured in IMDM
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(Gibco, USA); CAPAN-2 and PANC1 cells were cultured in high-glucose DMEM
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(Gibco, USA); and SW1990 cells were cultured in Leibovitz’s L-15 Medium (Gibco,
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USA) supplemented with 10% of fetal bovine serum at 37°C and 5% CO2 in a
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humidified atmosphere.
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Transient transfection of siRNA and stable transfection of shRNA were performed
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according to the manufacturers’ protocols as described elsewhere [12].
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2.6. Cell viability and a plate clone formation assay
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Cell viability was measured at 24, 48, and 72 h after respective treatments with Cell
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Counting Kit-8 (CCK8) (DOJINDO, Kumamoto, Japan) according to the
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manufacturer’s instructions. Absorbance values were measured at the wavelength of
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450 nm as representation of cell viability.
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For the plate clone formation assay, 600 cells per well were seeded in a 6-well plate
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and cultured for 12 days. The culture medium was changed every 4 days. Then, the
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cells were fixed in 4% formaldehyde and stained with crystal violet. Cell clones were
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counted and analyzed.
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2.7. Construction of plasmids and a luciferase activity assay 8
ACCEPTED MANUSCRIPT Potential upstream promoter regions of LINC01133 were amplified by PCR and
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cloned into the pGL3.0-basic vector (Promega, Madison, WI). A series of
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progressively truncated promoters and two C/EBPβ-binding site mutant promoter
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fragments (Mut1: ATTGTGAAAC to ACCCATAGGA, Mut2: AGTTGCACCAG to
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ATCCATCAGGA) were amplified by PCR and cloned into the pGL3.0-basic vector.
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A dual luciferase reporter assay was carried out by means of the Dual-luciferase
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Reporter Assay System (Promega, Madison, WI) according to manufacturer’s
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instructions.
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2.8. The chromatin immunoprecipitation (ChIP) assay
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ChIP and subsequent PCR were conducted with the Magna ChIP™ Kit (Millipore,
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USA) according to manufacturers’ instructions. Chip grade primary antibodies against
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C/EBPβ (Abcam, UK) were used in the ChIP experiment, and a normal rabbit IgG
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(Santa Cruz Biotechnology, USA) served as a negative control.
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2.9. Animal experiments
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These experiments were performed as described previously [12]. To establish the
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subcutaneous xenograft models, SW-1990 and BXPC3 cells (107) resuspended in 150
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µL of PBS were subcutaneously injected into the right flank of nude mice, and the
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tumor volume was measured every 4 days by means of a caliper and calculated as
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length × width2/2. Thirty-two days after implantation, the mice were euthanized by
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cervical dislocation according to the protocol filed with the Guidance of Institutional
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Animal Care and Use Committee (IACUC) of Sun Yat-Sen University, and tumor
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xenografts were then excised, fixed, weighed, photographed, and stored. All the
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animal experiments were carried out with the approval of the Institutional Review
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Board of Sun Yat-Sen University (IACUC- DB-17-1008).
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2.10. Statistical analysis
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All the experiments were independently repeated at least three times. Statistical
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analysis was carried in the SPSS software 18.0 for Windows (SPSS Inc., Chicago, IL,
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USA). Data were expressed as mean ± standard deviation. The significance of
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differences between groups was estimated by the Student t test, Wilcoxon test, or Chi
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square test. The DFS and OS were determined by the Kaplan-Meier method, and the
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log-rank test was carried out to evaluate the inter-group differences. The cutoff points
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for C/EBPβ expression from TCGA database for drawing the Kaplan-Meier survival
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curves were determined in the X-tile software (Version 3.6.1, Yale University, New
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Haven, CT, USA). Heatmaps were built using R programming. Pearson correlation
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analyses were applied to investigate the correlation among LINC01133, CCNG1, and
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C/EBPβ expression levels. In all the statistical analyses, data with two-tailed p < 0.05
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were considered statistically significant.
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3.1. LINC01133 expression was upregulated in PDAC and correlated with a poor
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prognosis
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To identify lncRNAs aberrantly expressed in PDAC, we first analyzed all the
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differentially expressed lncRNAs within the three microarray datasets (GSE15471,
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GSE16515, and GSE32676) from the GEO database (Figure 1A). Seven lncRNA
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probes were consistent in all the three datasets (Figure 1B). LINC01133 was one of
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the most significantly upregulated lncRNAs among the three datasets (Figure 1C, all p
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< 0.05). Furthermore, LINC01133 expression levels were analyzed in a cohort of 49
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pairs of PDAC and adjacent non-cancerous tissues. The results showed that
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LINC01133 expression was significantly higher in PDAC tissues compared to
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adjacent non-cancerous tissues (Figure 1D, p < 0.01). Increased LINC01133 levels in
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PDAC positively correlated with tumor size (p = 0.043), T stage (p = 0.044), and
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TNM stage (p = 0.024). No correlation was observed between LINC01133 expression
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and other parameters, such as gender (p = 0.645) and age (p = 0.488; Table 1).
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To clarify the prognostic value of the seven lncRNA among PDAC patients, the
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relation between their expression and survival time was next investigated in TCGA
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database. As shown in Figure 1E, a significant difference in DFS and OS was
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observed between high and low LINC01133 expression groups (p = 0.0136 and p =
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0.0467, respectively). Moreover, the expression levels of lncRNA PVT1 and
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CTD-2377D24 were also associated with poorer prognosis among the patients
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(Supplemental Figure 1A and 1B). The correlation between LINC01133 expression
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analyzed next. Increased expression of LINC01133 in PDAC correlated with
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histological grade (p < 0.001), disease-free status (p = 0.004), and mutation count (p =
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0.026). On the other hand, LINC01133 expression did not correlate with other
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parameters, such as gender (p = 0.501) and age (p = 0.707; Table 2).
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3.2. The knockdown of LINC01133 inhibited proliferation of PDAC cells in vitro
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We next examined the expression of LINC01133 in 5 human PDAC cell lines and
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found that LINC01133 expression was higher in SW1990 and BXPC3 cells than in
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other PDAC cell lines (Figure 2A). To investigate the functional effects of
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LINC01133 in PDAC cells, LINC01133 expression was suppressed by siRNA
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transfection in the SW1990 and BXPC3 cell lines (Figure 2B). We next examined the
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effect of LINC01133 on the cell proliferative ability by CCK8 and colony formation
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assays. Downregulation of LINC01133 substantially reduced the rates of cell
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proliferation and colony formation of SW1990 and BXPC3 cells (Figure 2C and 2D).
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In addition, silencing LINC01133 significantly increased cell apoptosis in SW1990
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and BXPC3 cells (Supplemental Figure 2). But downregulation of LINC01133 did not
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affect the cell cycle, cell migration and invasive activity in SW1990 and BXPC3 cells
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(Supplemental Figure 3A and 3B).
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3.3. The knockdown of LINC01133 impaired PDAC tumorigenicity in vivo
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To test whether the level of LINC01133 expression could affect PDAC cell growth in 12
ACCEPTED MANUSCRIPT vivo, we constructed LINC01133 stable knockdown SW1990 and BXPC3 cell lines
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using a lentivirus carrying shRNA. LINC01133 knockdown cells and control cells
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were subcutaneously injected into BALB/c nude mice. The LINC01133-deficient
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tumors grew more slowly than did tumors in control groups in both SW1990 and
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BXPC3 xenograft models (Figure 3A). The mice were euthanized, and tumors were
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measured 32 days after the cell injection (Figure 3B). The tumor weight at the end of
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the experiment was markedly lower in the shLINC01133-transfected SW1990 and
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BXPC3 groups compared with the empty-vector group (Figure 3C, p < 0.01).
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LINC01133 knockdown efficiency was validated by quantitative PCR in LINC01133
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knockdown cell–derived tumors (Figure 3D). Moreover, IHC staining revealed that
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proliferation marker gene Ki67 was dramatically downregulated in LINC01133
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knockdown tumors (Figure 3E). These findings indicated that the knockdown of
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LINC01133 inhibits tumorigenesis in vivo.
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3.4. C/EBPβ positively regulated LINC01133 transcription by directly binding to the
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promoter of LINC01133
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Transcription factors are the most important regulators of transcription. To clarify the
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reason for LINC01133 overexpression in PDAC, we cloned the promoter of
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LINC01133 and constructed a series of sequentially truncated LINC01133 promoters
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in the region from position −2000 to 0 relative to the transcription start site (hg19
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chr1:159,931,008). Transcriptional activity analysis indicated that the transcriptionally
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ACCEPTED MANUSCRIPT active region was mainly located between positions −1000 and 0 (Figure 4A). Next,
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transcription factor prediction analysis was performed in online software JASPAR and
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with ChIP-seq data of the UCSC genome browser. Interestingly, we found two strong
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C/EBPβ-binding sites in the predicted promoter area of the LINC01133 gene (Figure
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4A). Mutation of each of the two individual C/EBPβ-binding sites significantly
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decreased the LINC01133 transcriptional activity in SW1990 and BXPC3 cells, and
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mutation of the two C/EBPβ-binding sites simultaneously decreased the LINC01133
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transcriptional activity in SW1990 and BXPC3 cells to a lower level (Figure 4B).
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Correlation analysis of the dataset from TCGA indicated that the expression levels of
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LINC01133 and C/EBPβ positively correlated (Figure 4C, p = 0.002). Moreover, the
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correlation was validated in 5 PDAC cell lines (Figure 4D, p = 0.0013). Therefore, we
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hypothesized that C/EBPβ may regulate the transcription of LINC01133.
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LINC01133 expression decreased in SW1990 and BXPC3 cells transfected with
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C/EBPβ siRNA as compared with the control cells. In contrast, LINC01133
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expression increased in CFPAC1 and PANC1 cells with C/EBPβ overexpression as
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compared with the control cells (Figure 4E). ChIP analysis of the LINC01133
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promoter with an antibody against C/EBPβ followed by PCR amplifying the two
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characterized C/EBPβ-binding sites—was performed to determine whether C/EBPβ
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binds to the LINC01133 promoter. The results showed that C/EBPβ directly bound to
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the two C/EBPβ-binding sites of the LINC01133 promoter in SW1990 and BXPC3
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cells (Figure 4F).
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To investigate whether C/EBPβ is also aberrantly expressed in PDAC, C/EBPβ
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normal tissues by IHC analysis. The results indicated that C/EBPβ expression was
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significantly higher in PDAC tissues compared with adjacent normal tissues (Figure
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4G, p < 0.01). To clarify the prognostic value of C/EBPβ in PDAC patients, the
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relation between C/EBPβ expression and survival time was then investigated among
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83 patients with PDAC. The high C/EBPβ expression group had poorer DFS and OS
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than did the group with low C/EBPβ expression (p = 0.0098 and p = 0.0192,
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respectively; Figure 4H). Similarly, it was found that the C/EBPβ level negatively
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correlated with DFS and OS periods among PDAC patients recruited from TCGA
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database (p = 0.0493 and p = 0.0019, respectively; Figure 4I).
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3.5. LINC01133 promoted PDAC proliferation by upregulating CCNG1
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To further illustrate the molecular mechanisms underlying oncogenic effects of
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LINC01133 in PDAC, we conducted RNA-seq to analyze the global change of gene
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expression after depletion of LINC01133 by shRNA transfection in BXPC3 cells.
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Among all the differentially expressed genes, we found that the expression of cyclin
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G1 (CCNG1) was significantly decreased by shLINC01133 treatment (Figure 5A).
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We performed correlation analysis between the expression of LINC01133 and
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CCNG1 in TCGA database and detected a good positive correlation between
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LINC01133 and CCNG1 (Figure 5B, p = 0.022). After that, we confirmed that
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downregulation of LINC01133 led to a consistent decrease of CCNG1 mRNA and
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protein levels in SW1990 and BXPC3 cells (Figure 5C). Expression of CCNG1 was
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mRNA level (Figure 5D, all the p values <0.01) and protein level (Figure 5E, p <
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0.01). These findings indicated that the knockdown of LINC01133 suppressed the
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expression of CCNG1.
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To test whether CCNG1 is involved in the promotion of PDAC cell proliferation by
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LINC01133, we performed gain-of-function assays. We first used CCNG1 siRNA to
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knock down CCNG1 expression in SW1990 and BXPC3 cell lines; this knockdown
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was confirmed by qPCR and western blot analysis (Figure 6A, all the p < 0.01).
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Meanwhile, CCK8 and colony formation assays revealed that downregulation of
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CCNG1 inhibited proliferation of SW1990 and BXPC3 cells (Figure 6B and 6C).
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Rescue assays were performed to determine whether LINC01133 regulates PDAC cell
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proliferation by increasing CCNG1 expression (Supplemental Figure 4). CCK8 and
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colony formation assays indicated that overexpression of CCNG1 attenuated
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LINC01133-mediated inhibition of proliferation of SW1990 and BXPC3 cells (Figure
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6D and 6E). These data indicated that LINC01133 regulates proliferation of PDAC
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cells at least partially through the increase in CCNG1 expression.
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LINC01133 has been found to be aberrantly expressed in various cancers and to have
339
controversial functions. A recent report showed that LINC01133 is upregulated in
340
lung squamous cell cancer [13]. In addition, an oncogenic function of LINC01133 in
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non–small cell lung cancer was revealed by repressing KLF2, p21, and E-cadherin
342
transcription [14]. In human osteosarcoma, LINC01133 enhances the proliferation,
343
migration, and invasion of cancer cells by sponging miR-422a [15]. Nonetheless, the
344
expression of LINC01133 is significantly lower in colorectal cancer tissues; and
345
LINC01133 inhibits epithelial–mesenchymal transition and metastasis by interacting
346
with SRSF6 [16, 17]. Therefore, the expression and function of LINC01133 may vary
347
in a tissue- and organ-specific manner [18]. Our study for the first time shows that
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increased LINC01133 expression performs an oncogenic function in PDAC tumor
349
development. The knockdown of LINC01133 exerted tumor-suppressive effects by
350
impairing cell proliferation.
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The transcriptional regulation of LINC01133 is unclear, which is important for
353
developing therapeutic agents targeting LINC01133. Here, we found that C/EBPβ, a
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member of the C/EBP family of transcription factors, is responsible for the
355
transcription of LINC01133 by binding to the promoter of LINC01133 in PDAC cells
356
[19]. C/EBPβ has been found to regulate cell proliferation, differentiation, and
357
apoptosis in a variety of cancers [20-22]. Nonetheless, the prognostic significance and
358
function of C/EBPβ in human pancreatic cancer has not yet been illustrated. In our
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compared with adjacent normal tissues, and this upregulation correlates with shorter
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survival time. Overall, our study allows us to speculate that C/EBPβ may serve as a
362
novel therapeutic target in PDAC.
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Besides, our study revealed that the oncogenic mechanism of action of LINC01133 in
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human PDAC is different from that in other cancers. The oncogenic role of cyclin G1
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has been well documented, and its overexpression in a variety of human tumors has
367
been reported [23-25]. On the other hand, few studies detailed the function of CCNG1
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in pancreatic cancer. In this study, we revealed that the transcription of CCNG1 is
369
positively regulated by LINC01133, which promoted PDAC cell proliferation. All
370
these results suggest that CCNG1 may be a novel oncogene in PDAC and is regulated
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by LINC01133. To investigate the regulatory mechanism of LINC01133’s action on
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CCNG1, two bioinformatics websites Targetscan (http://www.targetscan.org/) and
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DIANA
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site%2Ftools) were used. We found that microRNA 1271 (miR-1271) contains
375
targeting sites of both LINC01133 and CCNG1 (data not shown). Therefore, we
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hypothesized that LINC01133 increases the transcription of CCNG1 by acting as a
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miR-1271 sponge; this model will be investigated in a future study.
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In conclusion, our study shows that LINC01133 expression is higher in human PDAC
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tissues compared to adjacent non-cancerous tissues, and this higher expression is 18
ACCEPTED MANUSCRIPT associated with poorer prognosis among the patients. LINC01133 is directly regulated
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by C/EBPβ and promotes cell proliferation in PDAC by upregulating CCNG1 (Figure
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6F). These findings may provide a clinically relevant rationale for developing a
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therapeutic strategy targeting C/EBPβ-LINC01133 axis for PDAC treatment.
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5. Acknowledgments
386
None
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6. Funding Sources
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This work was supported by the National Natural Science Foundation of China (grant
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numbers 81472261, 81572766), the Natural Science Foundation of Guangdong
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Province
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Development
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201604020044), the Guangdong Innovative and Entrepreneurial Research Team
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Program (grant number 2016ZT06S029) and the China Postdoctoral Science
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Foundation (grant number 2016M602588).
number of
Guangzhou,
the
Science
and
Guangdong,
China
Technology
(grant
number
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2015A030313032),
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(grant
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7. Ethics approval
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All procedures and data analyses in the patient were approved by the ethics committee
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of The First Affiliated Hospital of Sun Yat-sen University.
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ACCEPTED MANUSCRIPT Table1: Correlation between C/EBPβ characteristics in 83 PDAC patients Characteristics
CA19-9, kU/L
TBIL, µmol/L
TNM stage
Male
33
16
Female
21
13
≤60
28
＞60
26
≤5cm
39
＞5cm
15
≤37
12
＞37
42
≤ 34.4
17
>34.4
37
I
2
2
3
7
47
20
2
0
Positive
13
8
Negative
41
21
Positive
6
1
Negative
48
28
Positive
22
8
Negative
32
21
Positive
41
6
Negative
13
23
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0.872
0.810
19
1
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10
6
IV
0.043
23
0
III
*
6
2
I
0.488
2
20
II
Lymphatic metastasis
27
43
IV
0.645
11
8
III
expression
18
3
II
T stage
expression Low C/EBPβ
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Tumor size
C/EBPβ
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Age
Nerve invasion
clinicopathological
P-value*
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Vascular invasion
and
Number of patients High
Distant metastasis
expression
0.024
0.044
0.794
0.412
0.338
0.791
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P-value*
number of patients
Tumor stage
expression
Male
42
38
Female
46
51
≤60
28
26
＞60
60
T1/T2
T3/T4
Lymphatic metastasis
Negative
11
66
78
24
26
61
61
Negative
39
39
Positive
1
4
Ⅰ
14
7
Ⅱ
69
76
Ⅲ/Ⅳ
3
5
Neoplasm histologic
G1
25
6
grade
G2
37
58
G3/4
25
24
DiseaseFree
39
18
Recurred/
35
46
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TNM stage
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Distant metastasis
Disease Free statues
0.501
0.707
63
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Positive
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Age
Low LINC01133
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Gender
High LINC01133
0.058
0.812
0.193
0.207
<0.001
0.004
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Mutation count
44
41
≥50
21
42
0.026
Chi-square test.
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<50
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ACCEPTED MANUSCRIPT Figure legends Figure 1. LINC01133 is up-regulated and correlated with poor prognosis in human PDAC. (A) Heatmap showing differential expressed lncRNA in three GEO
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dataset. (B) Venn diagram representation of the overlap of 7 lncRNA that are differently expressed in all the three GEO dataset. (C) Relative expression of LINC01133 in the three gene expression profiles from GEO datasets. (D) Relative
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expression of LINC01133 in PDAC tissues (n=49) and non-tumor tissues (n=49) by
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qPCR. Results were presented as the log10-△CT value. (E) Correlation analysis of LINC01133 expression with disease-free survival and overall survival in PDAC patients from TCGA database. The P value was calculated by a log-rank test. HR：
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Hazard Ratio. All *p < 0.05, **p < 0.01.
Figure 2. Downregulation of LINC01133 expression inhibited PDAC cell proliferation in vitro. (A) Expression levels of LINC01133 in PDAC cell lines. (B)
(C)
Cell
viability
of
SW1990
and
BXPC3
cells
after
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Expression of LINC01133 in SW1990 and BXPC3 cells transfected with LINC01133
si-LINC01133-transfection. Data are presented as mean ± SD. (D) Colony–forming assays after si-LINC01133- transfection in SW1990 and BXPC3 cells. All *p < 0.05, **p < 0.01.
Figure 3. Knockdown LINC01133 impaired PDAC tumorigenicity in vivo. (A) Tumor growth curves after the injection of SW1990 and BXPC3 cells. Tumor volume
ACCEPTED MANUSCRIPT was calculated every 4 days. (B) The nude mice were sacrificed thirty-two days after the injection and tumors from respective groups were shown. (C) Tumor weight was measured after different treatments. N=6. (D) qPCR analysis of LINC01133
tumors with different treatments. All *p < 0.05, **p < 0.01.
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expression in tumor tissues from indicated group. N=6. (E) IHC staining of Ki67 in
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Figure 4. C/EBPβ directly regulated the expression of LINC01133 in PDAC cells.
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(A) Diagram of LINC01133 promoter, predicted two C/EBPβ binding sites and two designed mutation sequence of C/EBPβ binding sites were shown. Luciferase reporter assay for SW1990 and PANC1 cells transfected with reporter plasmids containing truncated LINC01133 promoters. (B) Luciferase reporter assay for wild type and
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C/EBPβ binding site mutant LINC01133 promoters’ (-1000bp ~0bp) activity in SW1990 and PANC1 cells. (C) Analysis of the relationship between LINC01133 expression and C/EBPβ expression levels in TCGA database. (D) Analysis of the
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relationship between LINC01133 expression and C/EBPβ expression levels in 5
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PDAC cell lines. (E) C/EBPβ and LINC01133 mRNA expression after C/EBPβ siRNA transfection in SW1990 and BXPC3 cells or C/EBPβ plasmid transfection in CFPAC1 and PANC1 cells. (F) The two C/EBPβ binding sites in the LINC01133 gene promoter were detected in the chromatin sample immunoprecipitated from SW1990 and BXPC3 cells using an antibody against C/EBPβ. (G) The protein expression of C/EBPβ in PDAC tissues and adjacent non-cancerous tissues were determined by immunohistochemistry. Box plots were presented for comparing
ACCEPTED MANUSCRIPT C/EBPβ expression between 83 PDAC tissues and adjacent non-cancerous tissues. (H and I) Analysis of disease-free survival and overall survival in 83 PDAC patients (H) and PDAC patients from TCGA database (I) by LINC01133 expression. The P value
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was calculated by a log-rank test. All *p < 0.05, **p < 0.01.
Figure 5. LINC01133 regulated the expression of CCNG1. (A) Heatmap showing
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the expression changes of BXPC3 cells after transfection of LINC01133 shRNA and
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control shRNA. Gene expression is shown as RPKM (Reads Per Kilobase per Million mapped reads) after normalization. (B) Analysis of the relationship between LINC01133 expression and CCNG1 expression levels in TCGA database. (C) Downregulation of LINC01133 expression with LINC01133 shRNAs downregulated
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CCNG1 mRNA and protein expression in SW1990 and BXPC3 cells. (D) qPCR analysis of CCNG1 mRNA expression in nude mice tumor tissues after different treatment. N=6. (E) IHC staining analysis of CCNG1 protein expression of nude mice
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tumors tissues after different treatment. N=6. All *p < 0.05, **p < 0.01.
Figure 6. LINC01133 function as oncogene by upregulating CCNG1 expression in PDAC cells. (A) BXPC3 cells or SW1990 cells were transfected with CCNG1 siRNA. Relative mRNA and protein expression of CCNG1 were detected by RT-qPCR and Western blot. (B) CCK8 or (C) colony-forming assays were used to determine the cell viability of BXPC3 cells or SW1990 cells co-transfected with CCNG1 siRNA. (D) CCK8 or (E) colony-forming assays were used to determine the
ACCEPTED MANUSCRIPT cell viability of BXPC3 cells or SW1990 cells co-transfected with indicated siRNA or/and plasmid. (F) Schematic illustration of our working model by which C/EBPβ regulated the expression of LINC01133 by directly binding to its promoter region and
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subsequently activating CCNG1 expression. Data represent the mean±SD. All *p <
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0.05, **p < 0.01.
ACCEPTED MANUSCRIPT LINC01133 was upregulated in pancreatic ductal adenocarcinoma (PDAC) and associated with poor prognosis. C/EBPβ regulated the transcription of LINC01133 and was related to the
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carcinogenesis of PDAC. LINC01133 promotes PDAC proliferation in a CCNG1-dependent manner.
We identified C/EBPβ-LINC01133 axis as a potential drug target and biomarker for
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PDAC.
S0304-3835(18)30153-8
DOI:
10.1016/j.canlet.2018.02.020
Reference:
CAN 13770
To appear in:
Cancer Letters
Received Date: 8 January 2018 Revised Date:
11 February 2018
Accepted Date: 12 February 2018
Please cite this article as: C.-S. Huang, J. Chu, X.-X. Zhu, J.-H. Li, X.-T. Huang, J.-P. Cai, W. Zhao, X.Y. Yin, The C/EBPβ-LINC01133 axis promotes cell proliferation in pancreatic ductal adenocarcinoma through upregulation of CCNG1, Cancer Letters (2018), doi: 10.1016/j.canlet.2018.02.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Abstract Long non-coding RNAs (lncRNAs) are emerging as important regulators and prognostic markers of multiple cancers. Our aim was to determine functional
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involvement of lncRNAs in pancreatic ductal adenocarcinoma (PDAC). In this study, we report that LINC01133 expression is higher in PDAC tissues compared to adjacent non-cancerous tissues, and this overexpression is associated with poorer prognosis
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among the patients. In vitro, a knockdown of LINC01133 substantially decreased
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PDAC cell proliferation. Tumorigenicity of PDAC cells with the LINC01133 knockdown was significantly impaired in a xenograft model assay. Moreover, we determined that CCAAT/enhancer-binding protein β (C/EBPβ) positively regulates LINC01133 expression by binding to the response elements within the LINC01133
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promoter. Higher expression of C/EBPβ was observed in PDAC tissues, and this overexpression was also associated with the poorer prognosis. Furthermore, the LINC01133 knockdown decreased cyclin G1 (CCNG1) expression. Overexpression
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of CCNG1 attenuated the LINC01133 silencing–induced impairment of proliferation
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in PDAC cells. In summary, our findings revealed that the C/EBPβ-LINC01133 axis performs an oncogenic function in PDAC by activating CCNG1, which may serve as a prognostic biomarker or a therapeutic target in PDAC.
ACCEPTED MANUSCRIPT The C/EBPβ-LINC01133 axis promotes cell proliferation in pancreatic ductal
2
adenocarcinoma through upregulation of CCNG1
3
Chen-Song Huang1#, Junjun Chu2#, Xiao-Xu Zhu1, Jian-Hui Li1, Xi-Tai Huang1,
4
Jian-Peng Cai1, Wei Zhao2*, Xiao-Yu Yin1*
5
1
6
Yat-sen University, Guangzhou 510080, China
7
2
8
# These authors contributed equally to this work.
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1
Department of Pancreato-Biliary Surgery, The First Affiliated Hospital of Sun
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Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China.
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Abbreviations: LncRNAs, Long non-coding RNAs; PDAC, pancreatic ductal
11
adenocarcinoma; C/EBPβ, CCAAT/enhancer-binding protein β; CCNG1, cyclin
12
protein G1; DFS, disease-free survival; OS, overall survival; siRNA, small interfering
13
RNA; qRT-PCR, quantitative real-time PCR; ChIP, chromatin immunoprecipitation;
14
GEO, Gene Expression Omnibus; PDAC, pancreatic ductal adenocarcinoma; shRNA,
15
short hairpin RNA; TCGA, The Cancer Genome Atlas
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*Correspondence
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Yin XY (email: [email protected])
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Zhao W (email: [email protected])
20
1
ACCEPTED MANUSCRIPT Abstract
22
Long non-coding RNAs (lncRNAs) are emerging as important regulators and
23
prognostic markers of multiple cancers. Our aim was to determine functional
24
involvement of lncRNAs in pancreatic ductal adenocarcinoma (PDAC). In this study,
25
we report that LINC01133 expression is higher in PDAC tissues compared to adjacent
26
non-cancerous tissues, and this overexpression is associated with poorer prognosis
27
among the patients. In vitro, a knockdown of LINC01133 substantially decreased
28
PDAC cell proliferation. Tumorigenicity of PDAC cells with the LINC01133
29
knockdown was significantly impaired in a xenograft model assay. Moreover, we
30
determined that CCAAT/enhancer-binding protein β (C/EBPβ) positively regulates
31
LINC01133 expression by binding to the response elements within the LINC01133
32
promoter. Higher expression of C/EBPβ was observed in PDAC tissues, and this
33
overexpression was also associated with the poorer prognosis. Furthermore, the
34
LINC01133 knockdown decreased cyclin G1 (CCNG1) expression. Overexpression
35
of CCNG1 attenuated the LINC01133 silencing–induced impairment of proliferation
36
in PDAC cells. In summary, our findings revealed that the C/EBPβ-LINC01133 axis
37
performs an oncogenic function in PDAC by activating CCNG1, which may serve as
38
a prognostic biomarker or a therapeutic target in PDAC.
39
Keywords: LINC01133; CCAAT/enhancer-binding protein β; Cyclin G1; Pancreatic
40
ductal adenocarcinoma; PDAC prognostic biomarker.
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ACCEPTED MANUSCRIPT 1. Introduction
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Pancreatic ductal adenocarcinoma (PDAC), accounting for more than 90% of
44
pancreatic cancer cases, is the fourth leading cause of death due to cancer in the
45
United States [1]. Only 15–20% of patients with pancreatic cancer have a diagnosis of
46
resectable disease. Patients with locally advanced cancer or metastasis have poor
47
prognosis [2,3]. Even though a large number of signaling pathways, growth factors,
48
oncogenes, and tumor suppressor genes have been found to participate in the initiation
49
and progression of pancreatic cancer [4], few of them are known to be useful for the
50
treatment of pancreatic cancer [5]. Therefore, identification of key regulators that
51
control PDAC carcinogenesis and progression is critically important for developing
52
more effective diagnostics and therapeutics.
53
Accumulating evidence reveals that long non-coding RNAs (lncRNAs) play
54
important roles in the carcinogenesis and progression of cancer. LncRNA GClnc1
55
performs a tumorigenic function by recruiting the WDR5 and KAT2A complex and by
56
modifying the transcription of SOD2 in human gastric cancer [6]. LncRNA ATB
57
upregulates ZEB1 and ZEB2 by competitively binding to members of the
58
microRNA-200 (miR-200) family and then induces epithelial–mesenchymal transition
59
and invasiveness of hepatocellular carcinoma [7]. Recently, another study showed that
60
LINC00673 reinforces the interaction of PTPN11 with PRPF19 and promotes
61
PTPN11 degradation through ubiquitination, which causes diminished SRC-ERK
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oncogenic signaling and enhances activation of the STAT1-dependent antitumor
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response [8]. Although several published studies have shown the involvement of
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addition, the mechanism of lncRNAs’ action on PDAC carcinogenesis needs to be
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further teased out.
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In the present study, we investigated the pathological role of LINC01133 in human
68
PDAC. LINC01133 levels were found to be remarkably upregulated in human PDAC
69
tumor tissues compared to adjacent non-cancerous tissues; this upregulation positively
70
correlated with the poor prognosis and shorter survival time. Both in vivo and in vitro
71
experiments revealed that LINC01133 promoted PDAC cell proliferation.
72
CCAAT/enhancer-binding protein β (C/EBPβ), a regulator of cell proliferation,
73
controls the expression of LINC01133 by directly binding to its promoter.
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LINC01133 next activates the transcription of cyclin protein G1 (CCNG1) and
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induces the proliferation and growth of cancer cells. Thus, our study revealed that the
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C/EBPβ-LINC01133 axis may act as a prognostic biomarker or a novel therapeutic
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target in PDAC.
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2.1. Patients’ specimens
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A total of 132 histologically proved PDAC patients, who underwent surgical resection
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of PDAC at the First Affiliated Hospital of Sun Yat-sen University (Guangzhou,
83
China), were recruited into the study. Among them, 83 PDAC patients, who
84
underwent pancreatectomy between January 2008 and December 2013, were recruited
85
for the clinicopathological and prognostic analysis. The inclusion criteria were as
86
follows: (1) undergoing curative resection; (2) receiving no preoperative
87
chemotherapy; (3) the absence of distant metastasis; (4) surviving longer than 30 days
88
after operation; (5) having the integrated clinicopathological data and follow-up data;
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(6) availability of a tumor tissue specimen. The clinicopathological characteristics are
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shown in Supplementary Table 1. In the remaining 49 patients who underwent
91
pancreatectomy between January 2015 and May 2017, tumorous and adjacent
92
non-cancerous tissues were obtained, snap-frozen instantly in liquid nitrogen, and
93
stored at −80°C until RNA extraction and quantitative real-time PCR for analysis of
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LINC01133 expression. This study’s protocol was approved by the Ethics Committee
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of the First Affiliated Hospital of Sun Yat-sen University. Written informed consent
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was obtained from each patient.
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2.2. Extraction and processing of Gene Expression Omnibus (GEO) and The Cancer
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Genome Atlas (TCGA) data
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Three Affymetrix® Human Genome U133-plus2 microarray datasets (GSE15471, 5
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the GEO database (https://www.ncbi.nlm.nih.gov/gds/). The array data of GSE15471
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[9] consisted of 36 PDAC tissue samples and 36 adjacent non-cancerous tissue
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samples. The array data of GSE16515 [10] included 36 PDAC tissue samples and 16
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adjacent non-cancerous tissue samples. The dataset of GSE32676 [11] included 25
106
PDAC tissue samples and 7 adjacent non-cancerous tissue samples. The .cel files of
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each sample were downloaded and analyzed using the “affy” package in R 3.4.1. A
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total of 3989 probes have been selected and annotated as lncRNAs. The expression
109
values of all those probes were extracted from the normalized expression data of the
110
whole array. Then, lncRNA probes differentially expressed between PDAC tissue and
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adjacent non-cancerous tissue were determined by an empirical Bayes statistics
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method of the “limma” package in R 3.4.1. The representative probe of LINC01133 is
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239370_at.
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TCGA data on LINC01133, C/EBPβ, and CCNG1 expression in PDAC tissues and
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clinical
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(https://tcga-data.nci.nih.gov/tcga/). LINC01133 expression in PDAC and its
118
correlations with clinical parameters of patients with PDAC were analyzed. The
119
Kaplan-Meier survival curves were constructed to examine the impact of the
120
LINC01133 gene on the disease-free survival (DFS) and overall survival (OS) of
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PDAC patients.
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PDAC tissues of 83 patients obtained from the Department of Pathology of the First
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Affiliated Hospital of Sun Yat-sen University and PDAC tumors from respective
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groups of xenograft nude mice were paraffin-embedded for IHC staining. This
127
staining was performed as described previously [12]. Two experienced pathologists
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independently scored the stained tissues according to both the area of positive staining
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and the intensity of staining. Cutoff values were chosen on the basis of heterogeneity
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measurement by the log-rank test with respect to OS and DFS.
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2.4. Reagents & antibodies
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Small interfering RNAs (siRNAs) targeting human LINC01133, C/EBPβ, or CCNG1
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and non-targeting control siRNA were purchased (Genepharma, Suzhou, China). The
135
pReceiver-M98-C/EBPβ overexpression plasmid and empty vector pReceiver-M98
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were purchased from Genecopoeia (Rockville, MD). Human LINC01133 small
137
hairpin RNA (shRNA) was ligated into the UUP vector to construct the LINC01133
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shLINC01133 plasmid. Human CCNG1 cDNA was ligated into the pcDNA3.1 vector
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to construct the pcDNA3.1-CCNG1 overexpression plasmid. The following
140
antibodies against the indicated proteins were employed in this study: a rabbit
141
anti-human C/EBPβ antibody [Abcam, UK], rabbit anti-human Ki-67 antibody
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[ProteinTech Group, USA], and rabbit anti-human CCNG1 antibody [ProteinTech
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Group, USA]. The sequences of primers, probes, shRNA, and siRNA used for the
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experiments in this study are listed in Supplementary Table 2.
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2.5. Cell culture and transfection
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Four PDAC cell lines (BXPC3, CFPAC1, PANC1, and SW1990) were purchased 7
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Academy of Sciences (Shanghai, China). PDAC cell line CAPAN-2 was acquired
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from the American Type Culture Collection (Manassas, VA, USA). BXPC3 cells
151
were cultured in RPMI 1640 (Gibco, USA); CFPAC1 cells were cultured in IMDM
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(Gibco, USA); CAPAN-2 and PANC1 cells were cultured in high-glucose DMEM
153
(Gibco, USA); and SW1990 cells were cultured in Leibovitz’s L-15 Medium (Gibco,
154
USA) supplemented with 10% of fetal bovine serum at 37°C and 5% CO2 in a
155
humidified atmosphere.
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Transient transfection of siRNA and stable transfection of shRNA were performed
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according to the manufacturers’ protocols as described elsewhere [12].
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2.6. Cell viability and a plate clone formation assay
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Cell viability was measured at 24, 48, and 72 h after respective treatments with Cell
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Counting Kit-8 (CCK8) (DOJINDO, Kumamoto, Japan) according to the
162
manufacturer’s instructions. Absorbance values were measured at the wavelength of
163
450 nm as representation of cell viability.
164
For the plate clone formation assay, 600 cells per well were seeded in a 6-well plate
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and cultured for 12 days. The culture medium was changed every 4 days. Then, the
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cells were fixed in 4% formaldehyde and stained with crystal violet. Cell clones were
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counted and analyzed.
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2.7. Construction of plasmids and a luciferase activity assay 8
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cloned into the pGL3.0-basic vector (Promega, Madison, WI). A series of
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progressively truncated promoters and two C/EBPβ-binding site mutant promoter
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fragments (Mut1: ATTGTGAAAC to ACCCATAGGA, Mut2: AGTTGCACCAG to
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ATCCATCAGGA) were amplified by PCR and cloned into the pGL3.0-basic vector.
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A dual luciferase reporter assay was carried out by means of the Dual-luciferase
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Reporter Assay System (Promega, Madison, WI) according to manufacturer’s
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instructions.
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2.8. The chromatin immunoprecipitation (ChIP) assay
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ChIP and subsequent PCR were conducted with the Magna ChIP™ Kit (Millipore,
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USA) according to manufacturers’ instructions. Chip grade primary antibodies against
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C/EBPβ (Abcam, UK) were used in the ChIP experiment, and a normal rabbit IgG
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(Santa Cruz Biotechnology, USA) served as a negative control.
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These experiments were performed as described previously [12]. To establish the
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subcutaneous xenograft models, SW-1990 and BXPC3 cells (107) resuspended in 150
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µL of PBS were subcutaneously injected into the right flank of nude mice, and the
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tumor volume was measured every 4 days by means of a caliper and calculated as
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length × width2/2. Thirty-two days after implantation, the mice were euthanized by
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cervical dislocation according to the protocol filed with the Guidance of Institutional
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Animal Care and Use Committee (IACUC) of Sun Yat-Sen University, and tumor
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xenografts were then excised, fixed, weighed, photographed, and stored. All the
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animal experiments were carried out with the approval of the Institutional Review
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Board of Sun Yat-Sen University (IACUC- DB-17-1008).
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2.10. Statistical analysis
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All the experiments were independently repeated at least three times. Statistical
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analysis was carried in the SPSS software 18.0 for Windows (SPSS Inc., Chicago, IL,
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USA). Data were expressed as mean ± standard deviation. The significance of
201
differences between groups was estimated by the Student t test, Wilcoxon test, or Chi
202
square test. The DFS and OS were determined by the Kaplan-Meier method, and the
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log-rank test was carried out to evaluate the inter-group differences. The cutoff points
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for C/EBPβ expression from TCGA database for drawing the Kaplan-Meier survival
205
curves were determined in the X-tile software (Version 3.6.1, Yale University, New
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Haven, CT, USA). Heatmaps were built using R programming. Pearson correlation
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analyses were applied to investigate the correlation among LINC01133, CCNG1, and
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C/EBPβ expression levels. In all the statistical analyses, data with two-tailed p < 0.05
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were considered statistically significant.
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3.1. LINC01133 expression was upregulated in PDAC and correlated with a poor
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prognosis
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To identify lncRNAs aberrantly expressed in PDAC, we first analyzed all the
215
differentially expressed lncRNAs within the three microarray datasets (GSE15471,
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GSE16515, and GSE32676) from the GEO database (Figure 1A). Seven lncRNA
217
probes were consistent in all the three datasets (Figure 1B). LINC01133 was one of
218
the most significantly upregulated lncRNAs among the three datasets (Figure 1C, all p
219
< 0.05). Furthermore, LINC01133 expression levels were analyzed in a cohort of 49
220
pairs of PDAC and adjacent non-cancerous tissues. The results showed that
221
LINC01133 expression was significantly higher in PDAC tissues compared to
222
adjacent non-cancerous tissues (Figure 1D, p < 0.01). Increased LINC01133 levels in
223
PDAC positively correlated with tumor size (p = 0.043), T stage (p = 0.044), and
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TNM stage (p = 0.024). No correlation was observed between LINC01133 expression
225
and other parameters, such as gender (p = 0.645) and age (p = 0.488; Table 1).
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To clarify the prognostic value of the seven lncRNA among PDAC patients, the
227
relation between their expression and survival time was next investigated in TCGA
228
database. As shown in Figure 1E, a significant difference in DFS and OS was
229
observed between high and low LINC01133 expression groups (p = 0.0136 and p =
230
0.0467, respectively). Moreover, the expression levels of lncRNA PVT1 and
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CTD-2377D24 were also associated with poorer prognosis among the patients
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(Supplemental Figure 1A and 1B). The correlation between LINC01133 expression
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analyzed next. Increased expression of LINC01133 in PDAC correlated with
235
histological grade (p < 0.001), disease-free status (p = 0.004), and mutation count (p =
236
0.026). On the other hand, LINC01133 expression did not correlate with other
237
parameters, such as gender (p = 0.501) and age (p = 0.707; Table 2).
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3.2. The knockdown of LINC01133 inhibited proliferation of PDAC cells in vitro
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We next examined the expression of LINC01133 in 5 human PDAC cell lines and
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found that LINC01133 expression was higher in SW1990 and BXPC3 cells than in
242
other PDAC cell lines (Figure 2A). To investigate the functional effects of
243
LINC01133 in PDAC cells, LINC01133 expression was suppressed by siRNA
244
transfection in the SW1990 and BXPC3 cell lines (Figure 2B). We next examined the
245
effect of LINC01133 on the cell proliferative ability by CCK8 and colony formation
246
assays. Downregulation of LINC01133 substantially reduced the rates of cell
247
proliferation and colony formation of SW1990 and BXPC3 cells (Figure 2C and 2D).
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In addition, silencing LINC01133 significantly increased cell apoptosis in SW1990
249
and BXPC3 cells (Supplemental Figure 2). But downregulation of LINC01133 did not
250
affect the cell cycle, cell migration and invasive activity in SW1990 and BXPC3 cells
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(Supplemental Figure 3A and 3B).
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3.3. The knockdown of LINC01133 impaired PDAC tumorigenicity in vivo
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To test whether the level of LINC01133 expression could affect PDAC cell growth in 12
ACCEPTED MANUSCRIPT vivo, we constructed LINC01133 stable knockdown SW1990 and BXPC3 cell lines
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using a lentivirus carrying shRNA. LINC01133 knockdown cells and control cells
257
were subcutaneously injected into BALB/c nude mice. The LINC01133-deficient
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tumors grew more slowly than did tumors in control groups in both SW1990 and
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BXPC3 xenograft models (Figure 3A). The mice were euthanized, and tumors were
260
measured 32 days after the cell injection (Figure 3B). The tumor weight at the end of
261
the experiment was markedly lower in the shLINC01133-transfected SW1990 and
262
BXPC3 groups compared with the empty-vector group (Figure 3C, p < 0.01).
263
LINC01133 knockdown efficiency was validated by quantitative PCR in LINC01133
264
knockdown cell–derived tumors (Figure 3D). Moreover, IHC staining revealed that
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proliferation marker gene Ki67 was dramatically downregulated in LINC01133
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knockdown tumors (Figure 3E). These findings indicated that the knockdown of
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LINC01133 inhibits tumorigenesis in vivo.
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3.4. C/EBPβ positively regulated LINC01133 transcription by directly binding to the
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promoter of LINC01133
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Transcription factors are the most important regulators of transcription. To clarify the
272
reason for LINC01133 overexpression in PDAC, we cloned the promoter of
273
LINC01133 and constructed a series of sequentially truncated LINC01133 promoters
274
in the region from position −2000 to 0 relative to the transcription start site (hg19
275
chr1:159,931,008). Transcriptional activity analysis indicated that the transcriptionally
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transcription factor prediction analysis was performed in online software JASPAR and
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with ChIP-seq data of the UCSC genome browser. Interestingly, we found two strong
279
C/EBPβ-binding sites in the predicted promoter area of the LINC01133 gene (Figure
280
4A). Mutation of each of the two individual C/EBPβ-binding sites significantly
281
decreased the LINC01133 transcriptional activity in SW1990 and BXPC3 cells, and
282
mutation of the two C/EBPβ-binding sites simultaneously decreased the LINC01133
283
transcriptional activity in SW1990 and BXPC3 cells to a lower level (Figure 4B).
284
Correlation analysis of the dataset from TCGA indicated that the expression levels of
285
LINC01133 and C/EBPβ positively correlated (Figure 4C, p = 0.002). Moreover, the
286
correlation was validated in 5 PDAC cell lines (Figure 4D, p = 0.0013). Therefore, we
287
hypothesized that C/EBPβ may regulate the transcription of LINC01133.
288
LINC01133 expression decreased in SW1990 and BXPC3 cells transfected with
289
C/EBPβ siRNA as compared with the control cells. In contrast, LINC01133
290
expression increased in CFPAC1 and PANC1 cells with C/EBPβ overexpression as
291
compared with the control cells (Figure 4E). ChIP analysis of the LINC01133
292
promoter with an antibody against C/EBPβ followed by PCR amplifying the two
293
characterized C/EBPβ-binding sites—was performed to determine whether C/EBPβ
294
binds to the LINC01133 promoter. The results showed that C/EBPβ directly bound to
295
the two C/EBPβ-binding sites of the LINC01133 promoter in SW1990 and BXPC3
296
cells (Figure 4F).
297
To investigate whether C/EBPβ is also aberrantly expressed in PDAC, C/EBPβ
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normal tissues by IHC analysis. The results indicated that C/EBPβ expression was
300
significantly higher in PDAC tissues compared with adjacent normal tissues (Figure
301
4G, p < 0.01). To clarify the prognostic value of C/EBPβ in PDAC patients, the
302
relation between C/EBPβ expression and survival time was then investigated among
303
83 patients with PDAC. The high C/EBPβ expression group had poorer DFS and OS
304
than did the group with low C/EBPβ expression (p = 0.0098 and p = 0.0192,
305
respectively; Figure 4H). Similarly, it was found that the C/EBPβ level negatively
306
correlated with DFS and OS periods among PDAC patients recruited from TCGA
307
database (p = 0.0493 and p = 0.0019, respectively; Figure 4I).
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3.5. LINC01133 promoted PDAC proliferation by upregulating CCNG1
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To further illustrate the molecular mechanisms underlying oncogenic effects of
311
LINC01133 in PDAC, we conducted RNA-seq to analyze the global change of gene
312
expression after depletion of LINC01133 by shRNA transfection in BXPC3 cells.
313
Among all the differentially expressed genes, we found that the expression of cyclin
314
G1 (CCNG1) was significantly decreased by shLINC01133 treatment (Figure 5A).
315
We performed correlation analysis between the expression of LINC01133 and
316
CCNG1 in TCGA database and detected a good positive correlation between
317
LINC01133 and CCNG1 (Figure 5B, p = 0.022). After that, we confirmed that
318
downregulation of LINC01133 led to a consistent decrease of CCNG1 mRNA and
319
protein levels in SW1990 and BXPC3 cells (Figure 5C). Expression of CCNG1 was
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321
mRNA level (Figure 5D, all the p values <0.01) and protein level (Figure 5E, p <
322
0.01). These findings indicated that the knockdown of LINC01133 suppressed the
323
expression of CCNG1.
324
To test whether CCNG1 is involved in the promotion of PDAC cell proliferation by
325
LINC01133, we performed gain-of-function assays. We first used CCNG1 siRNA to
326
knock down CCNG1 expression in SW1990 and BXPC3 cell lines; this knockdown
327
was confirmed by qPCR and western blot analysis (Figure 6A, all the p < 0.01).
328
Meanwhile, CCK8 and colony formation assays revealed that downregulation of
329
CCNG1 inhibited proliferation of SW1990 and BXPC3 cells (Figure 6B and 6C).
330
Rescue assays were performed to determine whether LINC01133 regulates PDAC cell
331
proliferation by increasing CCNG1 expression (Supplemental Figure 4). CCK8 and
332
colony formation assays indicated that overexpression of CCNG1 attenuated
333
LINC01133-mediated inhibition of proliferation of SW1990 and BXPC3 cells (Figure
334
6D and 6E). These data indicated that LINC01133 regulates proliferation of PDAC
335
cells at least partially through the increase in CCNG1 expression.
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338
LINC01133 has been found to be aberrantly expressed in various cancers and to have
339
controversial functions. A recent report showed that LINC01133 is upregulated in
340
lung squamous cell cancer [13]. In addition, an oncogenic function of LINC01133 in
341
non–small cell lung cancer was revealed by repressing KLF2, p21, and E-cadherin
342
transcription [14]. In human osteosarcoma, LINC01133 enhances the proliferation,
343
migration, and invasion of cancer cells by sponging miR-422a [15]. Nonetheless, the
344
expression of LINC01133 is significantly lower in colorectal cancer tissues; and
345
LINC01133 inhibits epithelial–mesenchymal transition and metastasis by interacting
346
with SRSF6 [16, 17]. Therefore, the expression and function of LINC01133 may vary
347
in a tissue- and organ-specific manner [18]. Our study for the first time shows that
348
increased LINC01133 expression performs an oncogenic function in PDAC tumor
349
development. The knockdown of LINC01133 exerted tumor-suppressive effects by
350
impairing cell proliferation.
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The transcriptional regulation of LINC01133 is unclear, which is important for
353
developing therapeutic agents targeting LINC01133. Here, we found that C/EBPβ, a
354
member of the C/EBP family of transcription factors, is responsible for the
355
transcription of LINC01133 by binding to the promoter of LINC01133 in PDAC cells
356
[19]. C/EBPβ has been found to regulate cell proliferation, differentiation, and
357
apoptosis in a variety of cancers [20-22]. Nonetheless, the prognostic significance and
358
function of C/EBPβ in human pancreatic cancer has not yet been illustrated. In our
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360
compared with adjacent normal tissues, and this upregulation correlates with shorter
361
survival time. Overall, our study allows us to speculate that C/EBPβ may serve as a
362
novel therapeutic target in PDAC.
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Besides, our study revealed that the oncogenic mechanism of action of LINC01133 in
365
human PDAC is different from that in other cancers. The oncogenic role of cyclin G1
366
has been well documented, and its overexpression in a variety of human tumors has
367
been reported [23-25]. On the other hand, few studies detailed the function of CCNG1
368
in pancreatic cancer. In this study, we revealed that the transcription of CCNG1 is
369
positively regulated by LINC01133, which promoted PDAC cell proliferation. All
370
these results suggest that CCNG1 may be a novel oncogene in PDAC and is regulated
371
by LINC01133. To investigate the regulatory mechanism of LINC01133’s action on
372
CCNG1, two bioinformatics websites Targetscan (http://www.targetscan.org/) and
373
DIANA
374
site%2Ftools) were used. We found that microRNA 1271 (miR-1271) contains
375
targeting sites of both LINC01133 and CCNG1 (data not shown). Therefore, we
376
hypothesized that LINC01133 increases the transcription of CCNG1 by acting as a
377
miR-1271 sponge; this model will be investigated in a future study.
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In conclusion, our study shows that LINC01133 expression is higher in human PDAC
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tissues compared to adjacent non-cancerous tissues, and this higher expression is 18
ACCEPTED MANUSCRIPT associated with poorer prognosis among the patients. LINC01133 is directly regulated
382
by C/EBPβ and promotes cell proliferation in PDAC by upregulating CCNG1 (Figure
383
6F). These findings may provide a clinically relevant rationale for developing a
384
therapeutic strategy targeting C/EBPβ-LINC01133 axis for PDAC treatment.
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5. Acknowledgments
386
None
387
6. Funding Sources
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This work was supported by the National Natural Science Foundation of China (grant
390
numbers 81472261, 81572766), the Natural Science Foundation of Guangdong
391
Province
392
Development
393
201604020044), the Guangdong Innovative and Entrepreneurial Research Team
394
Program (grant number 2016ZT06S029) and the China Postdoctoral Science
395
Foundation (grant number 2016M602588).
number of
Guangzhou,
the
Science
and
Guangdong,
China
Technology
(grant
number
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2015A030313032),
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All procedures and data analyses in the patient were approved by the ethics committee
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of The First Affiliated Hospital of Sun Yat-sen University.
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ACCEPTED MANUSCRIPT Table1: Correlation between C/EBPβ characteristics in 83 PDAC patients Characteristics
CA19-9, kU/L
TBIL, µmol/L
TNM stage
Male
33
16
Female
21
13
≤60
28
＞60
26
≤5cm
39
＞5cm
15
≤37
12
＞37
42
≤ 34.4
17
>34.4
37
I
2
2
3
7
47
20
2
0
Positive
13
8
Negative
41
21
Positive
6
1
Negative
48
28
Positive
22
8
Negative
32
21
Positive
41
6
Negative
13
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0.872
0.810
19
1
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10
6
IV
0.043
23
0
III
*
6
2
I
0.488
2
20
II
Lymphatic metastasis
27
43
IV
0.645
11
8
III
expression
18
3
II
T stage
expression Low C/EBPβ
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Tumor size
C/EBPβ
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Age
Nerve invasion
clinicopathological
P-value*
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Vascular invasion
and
Number of patients High
Distant metastasis
expression
0.024
0.044
0.794
0.412
0.338
0.791
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P-value*
number of patients
Tumor stage
expression
Male
42
38
Female
46
51
≤60
28
26
＞60
60
T1/T2
T3/T4
Lymphatic metastasis
Negative
11
66
78
24
26
61
61
Negative
39
39
Positive
1
4
Ⅰ
14
7
Ⅱ
69
76
Ⅲ/Ⅳ
3
5
Neoplasm histologic
G1
25
6
grade
G2
37
58
G3/4
25
24
DiseaseFree
39
18
Recurred/
35
46
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TNM stage
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Distant metastasis
Disease Free statues
0.501
0.707
63
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Positive
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Low LINC01133
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High LINC01133
0.058
0.812
0.193
0.207
<0.001
0.004
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Mutation count
44
41
≥50
21
42
0.026
Chi-square test.
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<50
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ACCEPTED MANUSCRIPT Figure legends Figure 1. LINC01133 is up-regulated and correlated with poor prognosis in human PDAC. (A) Heatmap showing differential expressed lncRNA in three GEO
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dataset. (B) Venn diagram representation of the overlap of 7 lncRNA that are differently expressed in all the three GEO dataset. (C) Relative expression of LINC01133 in the three gene expression profiles from GEO datasets. (D) Relative
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expression of LINC01133 in PDAC tissues (n=49) and non-tumor tissues (n=49) by
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qPCR. Results were presented as the log10-△CT value. (E) Correlation analysis of LINC01133 expression with disease-free survival and overall survival in PDAC patients from TCGA database. The P value was calculated by a log-rank test. HR：
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Hazard Ratio. All *p < 0.05, **p < 0.01.
Figure 2. Downregulation of LINC01133 expression inhibited PDAC cell proliferation in vitro. (A) Expression levels of LINC01133 in PDAC cell lines. (B)
(C)
Cell
viability
of
SW1990
and
BXPC3
cells
after
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Expression of LINC01133 in SW1990 and BXPC3 cells transfected with LINC01133
si-LINC01133-transfection. Data are presented as mean ± SD. (D) Colony–forming assays after si-LINC01133- transfection in SW1990 and BXPC3 cells. All *p < 0.05, **p < 0.01.
Figure 3. Knockdown LINC01133 impaired PDAC tumorigenicity in vivo. (A) Tumor growth curves after the injection of SW1990 and BXPC3 cells. Tumor volume
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tumors with different treatments. All *p < 0.05, **p < 0.01.
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expression in tumor tissues from indicated group. N=6. (E) IHC staining of Ki67 in
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Figure 4. C/EBPβ directly regulated the expression of LINC01133 in PDAC cells.
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(A) Diagram of LINC01133 promoter, predicted two C/EBPβ binding sites and two designed mutation sequence of C/EBPβ binding sites were shown. Luciferase reporter assay for SW1990 and PANC1 cells transfected with reporter plasmids containing truncated LINC01133 promoters. (B) Luciferase reporter assay for wild type and
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C/EBPβ binding site mutant LINC01133 promoters’ (-1000bp ~0bp) activity in SW1990 and PANC1 cells. (C) Analysis of the relationship between LINC01133 expression and C/EBPβ expression levels in TCGA database. (D) Analysis of the
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relationship between LINC01133 expression and C/EBPβ expression levels in 5
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PDAC cell lines. (E) C/EBPβ and LINC01133 mRNA expression after C/EBPβ siRNA transfection in SW1990 and BXPC3 cells or C/EBPβ plasmid transfection in CFPAC1 and PANC1 cells. (F) The two C/EBPβ binding sites in the LINC01133 gene promoter were detected in the chromatin sample immunoprecipitated from SW1990 and BXPC3 cells using an antibody against C/EBPβ. (G) The protein expression of C/EBPβ in PDAC tissues and adjacent non-cancerous tissues were determined by immunohistochemistry. Box plots were presented for comparing
ACCEPTED MANUSCRIPT C/EBPβ expression between 83 PDAC tissues and adjacent non-cancerous tissues. (H and I) Analysis of disease-free survival and overall survival in 83 PDAC patients (H) and PDAC patients from TCGA database (I) by LINC01133 expression. The P value
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was calculated by a log-rank test. All *p < 0.05, **p < 0.01.
Figure 5. LINC01133 regulated the expression of CCNG1. (A) Heatmap showing
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the expression changes of BXPC3 cells after transfection of LINC01133 shRNA and
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control shRNA. Gene expression is shown as RPKM (Reads Per Kilobase per Million mapped reads) after normalization. (B) Analysis of the relationship between LINC01133 expression and CCNG1 expression levels in TCGA database. (C) Downregulation of LINC01133 expression with LINC01133 shRNAs downregulated
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CCNG1 mRNA and protein expression in SW1990 and BXPC3 cells. (D) qPCR analysis of CCNG1 mRNA expression in nude mice tumor tissues after different treatment. N=6. (E) IHC staining analysis of CCNG1 protein expression of nude mice
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tumors tissues after different treatment. N=6. All *p < 0.05, **p < 0.01.
Figure 6. LINC01133 function as oncogene by upregulating CCNG1 expression in PDAC cells. (A) BXPC3 cells or SW1990 cells were transfected with CCNG1 siRNA. Relative mRNA and protein expression of CCNG1 were detected by RT-qPCR and Western blot. (B) CCK8 or (C) colony-forming assays were used to determine the cell viability of BXPC3 cells or SW1990 cells co-transfected with CCNG1 siRNA. (D) CCK8 or (E) colony-forming assays were used to determine the
ACCEPTED MANUSCRIPT cell viability of BXPC3 cells or SW1990 cells co-transfected with indicated siRNA or/and plasmid. (F) Schematic illustration of our working model by which C/EBPβ regulated the expression of LINC01133 by directly binding to its promoter region and
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subsequently activating CCNG1 expression. Data represent the mean±SD. All *p <
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ACCEPTED MANUSCRIPT LINC01133 was upregulated in pancreatic ductal adenocarcinoma (PDAC) and associated with poor prognosis. C/EBPβ regulated the transcription of LINC01133 and was related to the
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carcinogenesis of PDAC. LINC01133 promotes PDAC proliferation in a CCNG1-dependent manner.
We identified C/EBPβ-LINC01133 axis as a potential drug target and biomarker for
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PDAC.