LincRNAFEZF1-AS1 represses p21 expression to promote gastric cancer proliferation through LSD1-Mediated H3K4me2 demethylation
© The Author(s). 2017
Received: 18 July 2016
Accepted: 13 January 2017
Published: 16 February 2017
Although the prognosis of gastric cancer patients have a favorable progression, there are some patients with unusual patterns of locoregional and systemic recurrence. Therefore, a better understanding of early molecular events of the disease is needed. Current evidences demonstrate that long noncoding RNAs (lncRNAs) may be an important class of functional regulators involved in human gastric cancers development. Our previous studies suggest that HOTAIR contributes to gastric cancer development, and the overexpression of HOTAIR predicts a poor prognosis. In this study, we investigated the characteristic of the LncRNA FEZF1-AS1 in gastric cancer.
QRT-PCR was used to detect the expression of FEZF1-AS1 in gastric cancer tissues and cells. MTT assays, clonogenic survival assays and nude mouse xenograft model were used to examine the tumorigenesis function of FEZF1-AS1 in vitro and in vivo. Bioinformatics analysis were used to select downstream target genes of FEZF1-AS1. Cell cycle analysis, ChIP, RIP,RNA Pulldown assays were examined to dissect molecular mechanisms.
In this study, we reported that FEZF1-AS1, a 2564 bp RNA, was overexpressed in gastric cancer, and upregulated FEZF1-AS1 expression indicated larger tumor size and higher clinical stage; additional higher expression of FEZF1-AS1 predicted poor prognosis. Further experiments revealed that knockdown FEZF1-AS1 significantly inhibited gastric cancer cells proliferation by inducing G1 arrest and apoptosis, whereas endogenous expression FEZF1-AS1 promoted cell growth. Additionally, RIP assay and RNA-pulldown assay evidenced that FEZF1-AS1 could epigenetically repress the expression of P21 via binding with LSD1, the first discovered demethylase. ChIP assays demonstrated that LSD1 could directly bind to the promoter of P21, inducing H3K4me2 demethylation.
In summary, these data demonstrated that FEZF1-AS1 could act as an “oncogene” for gastric cancer partly through suppressing P21 expression; FEZF1-AS1 may be served as a candidate prognostic biomarker and target for new therapies of gastric cancer patients.
KeywordsFEZF1-AS1 LSD1 H3K4me2 P21 Gastric cancer
Gastric cancer is the third leading cause of cancer-related deaths worldwide, and the poor prognosis of patients is largely due to the high frequency of tumor recurrence or metastasis within 24 months after surgical resection [1, 2]. To improve gastric cancer early diagnosis and targeted therapy, a better understanding of early molecular events of the disease is warranted. Cell proliferation is a pivotal characteristic of malignancy and a hallmark cancer capability . Dysregulation of all cycle is a vital reason for tumor cell proliferation. Moreover, the cell cycle regulation has come to be a promising therapeutic target, which suggests that discovery of novel proliferation related genes could lead to improve treatment of cancer [4, 5].
Recent integrative genomic studies have revealed that 98% of the human genome transcripts are non-coding RNA (ncRNA) with limited or no protein-coding capacity [6–8]. Long non-coding RNAs (lncRNAs), greater than 200 nt are important new members of the ncRNA family . Researchers have demonstrated that the aberrant lncRNAs expression involve in diverse human diseases, in particular cancers [10–12]. Such one is HOTAIR, lots of studies have shown that HOTAIR is overexpressed in colorectal cancer, pancreatic cancer, breast cancer, gastric cancer and gastrointestinal stromal tumors and is positively correlated with a poor clinical outcome [13–16]. Furthermore, lncRNA regulate drug resistance, for instance, H19 epigenetically inducted MDR1-associated drug resistance in human hepatocellular carcinoma cells . Recently, a study showed that nearly 76% of the GENCODE annotated lncRNAs was differentially expressed between gastric cancer and normal gastric tissue ; for example, HOTAIR and HOXA-AS2 were overexpressed in gastric cancer and indicated poor prognosis; however, a large number of lncRNAs have been uncharacterized [19–22].
Recently, mounting evidences showed that some lncRNAs epigenetically regulate gene expression by DNA methylation and histone modifications, which contain methylation, acetylation, phosphorylation et al. . Histone methylation is Histone H3/H4 on lysine different sites methylation or demethylation, which is regulated by histone methylases or demethylases. HOTAIR and ANRIL etc. could recruit and bind with the Polycomb complex PRC2 (EZH2, SUZ12 and EED), which enhances histone H3lysine-27 trimethylation, affecting chromatin compression tightness in suppressing gene expression [15, 24]. Lysine-specific demethylase 1(LSD1) is the first discovered demethylase, which demethylates mono— and di-methylated residues of lysine-4 on histone H3 (H3K4me1, H3K4me2 orH3K9me1) and results in transcriptional repression [25, 26]. In addition, LSD1 also activates transcription through demethylation of H3K9me2 . LSD1 is pivotal for mammalian tumorigenicity and progression in many type of cancers, moreover, LSD1 overexpression predict poor prognosis and aggressive tumor biology [28–31]. Many studies had shown LSD1 epigenetically regulate cell cycle related gene expression to affect G1/S phase arrest, contributing to cell proliferation [32–34].
FEZF1-AS1 is an lncRNA producing a 2564 bp transcript, located in chromosome 7. In this study, we demonstrated that FEZF1-AS1 was overexpressed in the tumor tissues than the paracancerous tissues; furthermore, overexpression of FEZF1-AS1 was observed in larger tumors, advanced gastric cancer and predicted poor DFS. Additional experiments revealed that FEZF1-AS1 knockdown significantly repressed proliferation both in vitro and vivo, and inhibited cells cycle progression by causing G1/S arrest. In addition, FEZF1-AS1 also recruited and bound to LSD1 to epigenetically repress downstream gene p21, thereby promoting proliferation in advanced stages of gastric cancer. By these efforts, we aim to propose a model for FEZF1-AS1-mediated cell proliferation in gastric cancer.
In this study, matched tumor tissues and adjacent non-tumor tissues were obtained from 82 gastric cancer patients at the Department of Surgical Oncology Jiangsu Province People’s Hospital, Nanjing Medical University from March 2011 to December 2011. Two pathologists evaluated all specimens according to the World Health Organization (WHO) guidelines and the pTNM Union for International Cancer Control (UICC) pathological staging criteria. No local or systemic treatments were administered to these patients before surgery. The tissues were immediately frozen in liquid nitrogen and stored at −80 °C until use. Informed consent was obtained from all patients. The Human Research Ethics Committee of Jiangsu Province People’s Hospital approved the study.
Total RNA extraction Quantitative real-time polymerase chain reaction
Total RNA was extracted from the cultured cells and frozen tissues using TRIzol reagent (Invitrogen, Karlsruhe, Germany) following the manufacturer’s protocol. Quantitative real-time polymerase chain reaction (PCR) was performed to detect FEZF1-AS1 and P21 using the PrimeScript RT Reagent Kit and SYBR Premix Ex Taq (TaKaRa, Dalian, China) according to the manufacturer’s instructions. The results were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The specific primers used are presented in Additional file 1: Table S1. The qPCR results were analyzed and expressed relative to the CT (threshold cycle) values and then converted to fold changes.2.0-fold change was considered significant.
The FEZF1-AS1 sequence was synthesized and subcloned into the pCDNA3.1 (Invitrogen, Shanghai, China) vector. Ectopic expression of FEZF1-AS1 was achieved via pCDNA-FEZF1-AS1 transfection, with an empty pCDNA3.1 vector used as a control. We also synthesised shRNA sequence targeted FEZF1-AS1. Si-FEZF1-AS1 sequence removed five bases of the 3 ‘end were converted to sh-FEZF1-AS1. After annealing of the complementary shRNA oligonucleotides, we cloned the annealed oligonucleotides into pENTR vector (sh-FEZF1-AS1) (Additional file 1: Table S1). The expression levels of FEZF1-AS1 were detected by qPCR.
The MGC-803 lines were cultured in RPMI 1640 medium containing 10% fetal bovine serum and incubated at 37 °C, 5% CO2, and saturated humidity. The SGC-7901 cells were cultured in DMEM medium containing 10% fetal bovine serum and incubated at 37 °C, 5% CO2, and saturated humidity. The AGS lines were cultured in F 12 medium containing 10% fetal bovine serum and incubated at 37 °C, 5% CO2, and saturated humidity. Cell growth was observed under an inverted microscope. Cells in the logarithmic growth phase were harvested for the experiments.
Plasmid vectors (pCDNA3.1-FEZF1-AS1 and pCDNA3.1) for transfection were prepared using DNA Midiprep or Midiprep kits (Qiagen, Hilden, Germany) and transfected into MGC-803cells. The si-FEZF1-AS1, sh-FEZF1-AS1, si-LSD1 or si-NC was transfected into AGS and SGC-7901 cells (Additional file 1: Table S1).
Cell cycle and apoptosis analysis
AGS and SGC-7901cells transiently transfected with si-FEZF1-AS1 or si-NC and MGC-803 transfected with pcDNA-FEZF1-AS1 or pcDNA-3.1, cells were analyzed by flow cytometry (FACScan; BD Biosciences) using CellQuest software (BD Biosciences).
MTT assay and clone formation
MTT assay and clone formation were used for evaluated cell viability and proliferation. Cell proliferation was documented following the manufacturer’s protocol every 24 h. For the colony formation assay, cells were seeded in a fresh six-well plate and maintained in media containing 10% FBS, replacing the medium every 4 days. After 14 days, methanol and stained with 0.1% crystal violet (Sigma-Aldrich) fixed cells and count clones.
Tumor formation assay in a nude mouse model
The male athymic BALB/c nude mice aged 5 weeks were maintained under specific pathogen-free conditions and manipulated according to protocols approved by the Shanghai Medical Experimental Animal Care Commission. A volume of 0.1 ml of suspended cells with sh-FEZF1-AS1 and pENTR vector (EV) was respectively subcutaneously injected into the posterior flank of each mouse. At 15 days post-injection, mice were euthanized and the primary tumors were excised, paraffin-embedded, formal infixed and performed H&E staining, immunostaining analysis for Ki-67 protein expression.
Western blotting analysis and antibodies
Cell lysates were prepared using RIPA protein extraction reagent (Beyotime, Beijing, China) supplemented with a protease inhibitor cocktail (Roche, CA, USA) and phenylmethylsulfonyl fluoride (Roche). GAPDH was used as a control. Antibodies (1:1000) against cyclin D1, CDK2, CDK4, CDK6 and P21were purchased from Abcam.
Subcellular fractionation location
The separation of nuclear and cytosolic fractions was performed using the PARIS Kit (Life Technologies) according to the manufacturer’s instructions.
Chromatin immunoprecipitation (ChIP)
We performed chromatin immunoprecipitation (ChIP) using the EZ ChIP™Chromatin Immunoprecipitation Kit for cell line samples (Millipore, Bedford, MA). Briefly, we sonicated the crosslinked chromatin DNA into 200- to 500-bp fragments. The chromatin was then immunoprecipitated using an anti-demethyl-histone H3 antibody and LSD1 (1:1000). Normal mouse IgG was used as the negative control. The primer sequences are listed in Additional file 1: Table S1. The antibodies for the ChIP assays of LSD1, H3K4 and H3K9 were obtained from Millipore. Quantification of the immunoprecipitated DNA was performed using qPCR with SYBR Green Mix (Takara). The ChIP data were calculated as a percentage relative to the input DNA using the equation 2[Input Ct- Target Ct] × 0.1 × 100.
We performed RNA immunoprecipitation (RIP) experiments using the Magna RIP™RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) according to the manufacturer’s instructions. The antibodies for the RIP assays of LSD1 were obtained from Abcam. The co-precipitated RNAs were detected by reverse-transcription PCR. The total RNAs were the input controls.
RNA pulldown assay
Biotin-labeled RNAs were transcribed in vitro with the Biotin RNA Labeling Mix (Roche Diagnostics) and T7 RNA polymerase (Roche Diagnostics), treated with RNase-free DNase I (Roche), and purified with an RNeasy Mini Kit (Qiagen, Valencia, CA). Next, 1 mg whole-cell lysates from SGC7901 cells was incubated with 3 μg of purified biotinylated transcripts for 1 h at 25 °C. Complexes were isolated with streptavidin agarose beads (Invitrogen). The beads were washed briefly three times and boiled in sodium dodecyl sulfate (SDS) buffer, and the retrieved protein was detected using the standard western blot technique.
Gene set enrichment analysis (GSEA) software was downloaded from Broad Institute (http://www.broadinstitute.org/gsea/index.jsp). Gene profiling data downstream FEZF1-AS1 were obtained from Gene Expression Omnibus (GEO) site (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE53137). Significantly enriched gene sets were identified, which produced a nominal P-value 0.05. UCSC Genome Browser (http://genome.ucsc.edu/cgi-bin/hgGateway) was used to analyze promoter regions.
The SPSS 17.0 statistical analysis software was used for the statistical analysis of the experimental data. The significance of differences between groups was estimated by Student’s t-test. The levels of FEZF1-AS1 in the gastric cancer patients were compared using the Mann–Whitney U test. The disease-free survival probability was analyzed using Kaplan-Meier methods and evaluated using the log-rank test. A p value less than 0.05 were considered significant.
FEZF1-AS1 expression levels in human gastric cancer tissue
FEZF1-AS1 upregulation associated with tumor size, stage and poor survival of gastric cancer patients
Correlation of the expression of FEZF1-AS1 with clinicopathologic features in gastric cancer
Transcription factor of promoter of FEZF1-AS1
Modulation of FEZF1-AS1expression in gastric cancer cells
To investigate the effect of FEZF1-AS1on the gastric cancer cells, we firstly examined the endogenous expression levels of FEZF1-AS1 in various cancer cell lines by qRT-PCR. As shown in Additional file 2: Figure S1A, of the five gastric cancer cell lines (SGC- 7901, BGC-823, MGC-803, AGS and HGC-27), SGC-7901 and AGS expressed higher levels of FEZF1-AS1 than the normal gastric epithelium cell line (GES-1); however, BGC-823 and MGC-803 expressed deficiency. Therefore, we chose SGC-7901 and AGS as loss of function experimental cell lines and MGC-803 as gain of function experimental cell line. The results showed that FEZF1-AS1 expression was effectively knocked down in AGS、SGC-7901and MGC-803cells by si-FEZF1-AS11# + 2#、si-FEZF1-AS12# + 3# and pcDNA-FEZF1-AS1 (Additional file 2: Figure S1B), which were subsequently used in the further experiments. The efficiency of the sh- FEZF1-AS1、si-LSD1、si-SP1 and pcDNA-SP1 was shown in Additional file 2: Figure S1C, D and E.
FEZF1-AS1 promoted gastric cancer cells proliferation in vitro and vivo
FEZF1-AS1 promoted proliferation of gastric cancer cells by inducing cell-cycle progress and reducing apoptosis in gastric cancer cells
FEZF1-AS1 downregulated P21 expression driving cell cycle
FEZF1-AS1epigenetically silenced P21 transcription through LSD1-Mediated H3K4me2 demethylation
In conclusion, these data indicated that FEZF1-AS1 recruit the LSD1 to repress P21 transcription via H3K4me2 modification.
Transcription factor SP1was involved in the upregulation of FEZF1-AS1
Over the past decades, mounting evidences have emphasized the emerging significance of lncRNAs in diverse human cancer, including gastric cancer [19, 37, 38]. Forthrtmore, a small part of the study has shown that lncRNA expression profiles is predicting cancer or discriminating between cancer subtypes. In fact, lncRNAs have an obvious merit of their relative tissue-specific expression and functional layout as transcriptional levels. lncRNAs may better reflect the biologic status of cancer cells. However, lncRNAs in gastric cancer are still an emerging field, only a few of lncRNAs have been characterized in gastric cancer tumorigenesis and should be further studied as predictive biomarkers. One of these lncRNAs is gastric adenocarcinoma predictive long intergenic noncoding RNA (GAPLINC), GAPLINC is overexpression and a predictive marker for metastasis and prognosis in gastric cancer . In this report, we found that the expression of another lncRNA, FEZF1-AS1, was significantly upregulated in gastric cancer tissues, and was correlated with poor prognosis. Furthermore, we have presented a study for the prediction of cancer/normal tissues and biomarkers using FEZF1-AS1 expression, suggesting that FEZF1-AS1 may be an independent clinical marker in gastric cancer diagnosis and prognosis.
The dysregulation of lncRNAs joins a wide variety of pathological processes, but the mechanisms of lncRNAs expression are not clear and further exploration is required. Transcription factor and epigenetic regulatory factors could manipulate the expression of lncRNAs [40, 41]. Here, through bioinformation analysis, we found that FEZF1-AS1promoter contained conserved SP1-binding site, which is a vital transcription factor in sustaining the “hall markers” of cancer . Accumulating data has revealed that SP1 is overexpressed in breast cancer and gastric cancer [43, 44]. Our findings evidenced that SP1 is a key factor in controlling FEZF1-AS1 expression. These results, along with those recent studies [45, 46], underline the role of transcription factors in regulating lncRNA transcription.
Additionally, our data demonstrated that knockdown FEZF1-AS1expression contributed to significant inhibition of cell proliferation both in vitro and in vivo, whereas exogenous expression FEZF1-AS1 led to cell growth. Downregulation FEZF1-AS1 expression caused G1 phase arrest and S phase reduction suppressing cell cycle progression. The G1–S transition in the cell cycle in mammalian cells is controlled by cyclins, cyclin-dependent kinases (CDKs) and their inhibitors, and the deregulation of CKIs is a common feature in tumor cells . P21, one of the most CKIs, is important checkpoints of P53 signaling pathway for G1/S transition by inhibiting the activity of kinases such as CyclinD/CDK4, CyclinD/CDK6 and CyclinE/CDK2 [48, 49], which plays multiple roles in inhibition cell proliferation in normal and cancer cells and was almost downregulated in many types of cancer. Notably, we found that P21 was remarkably upregulated upon FEZF1-AS1 knockdown. Our findings demonstrated that FEZF1-AS1 mediated gastric cancer cell proliferation promotion, which possibly also downregulated p21 expression.
A small number of functional lncRNAs have been well characterized, which can regulate gene expression at various levels, including chromatin modification, transcription and post-transcriptional processing. lncRNAs can act as molecular decoys binding and titrating away proteins or RNAs to indirectly exert biological functions in multiple kingdoms of life. HOTAIR is one of the most studied lncRNAs involved in chromatin modification, which can recruit PRC2 genome-wide to alter H3K27 methylation and gene expression patterns. lncRNA MALAT1 could bind to SFPQ to release PTBP2 from the SFPQ/PTBP2 complex and increase SFPQ-detached PTBP2 promoting CRC cell proliferation and migration . In addition, lncRNAs can recruit chromatin-modifying enzymes to target genes by acting as guides, either in cis (near the site of lncRNA production)  or in trans to distant target genes . In this study, the results of RNA and RNA-pull-down assays show that FEZF1-AS1 could bind with LSD1, the first discovered histone demethylase. LSD1 participate in development and differentiation regulation of chromatin remodeling and histone demethylation, which could specifically catalysed the demethylation of mono- and di-methylated histone H3 lysine 4(H3K4) and H3 lysine 9 (H3K9) through a redox process. More importantly, overexpression of LSD1 is involved in many pathological processes of cancer, such as proliferation, apoptosis and metastasis of various cancer cells [26, 28, 34]. S. Lim et al. reported  that knockdown LSD1 significantly reduced levels of H3K9me2 at the p21 locus regression cell proliferation through regulation of cell cycle. Our study demonstrated that knockdown FEZF1-AS1 led to enhance levels of H3K4me2 at the p21 promoter and a nearly unchanged H3K4me1 levels.
Bovine serum albumin
Cyclin-dependent kinase 2
Cyclin-dependent kinase 4
Cyclin-dependent kinase 6
Cyclin dependent kinase inhibitor
Dulbecco’s modified eagle medium
Epithelial-to- mesenchymal transition
Enhancer of zest homolog 2
Gene expression omnibus
Gene set enrichment analysis
Histone H3lysine-27 trimethylation
Histone H3lysine-4 mono-methylation
Histone H3lysine-4 di -methylation
Histone H3lysine-9 di -methylation
Hematoxylin and eosin
HOX antisense intergenic RNA
Long non-coding RNA
Lysine-specific demethylase 1
Polycomb group protein
Polycomb repressive complex 2
Quantitative real-time polymerase chain reaction
Receiver operating characteristic
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Short hairpin RNA
Small interfering RNA
Union for international cancer control
the World Health Organization
We would like to thank Dr. Yongqian Shu for his hard work in preparing the clinical specimens.
This work was supported by grants from the National Natural Science Foundation of China (No.81271699; No. 31401094).
Availability of data and materials
Please contact the corresponding author for all data requests.
Conception and design: WD, CLW,GZJ. Development of the methodology: MX, FY. Acquisition of data: YWL, RX. Analysis and interpretation of data: MS, RX. Writing revision of the manuscript: YWL. Administrative, technical, and material support: KL, MX. Study supervision: WD, CLW and GZJ. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
The research protocol was reviewed and approved by the Human Research Ethics Committee of Jiangsu Province People’s Hospital, and written informed consent was obtained from each patient included in the study.
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