Upregulation of lncRNA LINC00460 Facilitates GC Progression through Epigenetically Silencing CCNG2 by EZH2/LSD1 and Indicates Poor Outcomes

Non-protein-coding functional elements in the human genome in the postgenomic biology field have been drawing great attention in recent years. Thousands of long non-coding RNAs (lncRNAs) have been found to be expressed in various tumors. Yet only a small proportion of these lncRNAs have been well characterized. We have demonstrated that LINC00460 could affect cell proliferation through epigenetic regulation of KLF2 and CUL4A in human colorectal cancer. However, the clinical significance and biological role of LINC00460 in gastric cancer (GC) remain largely unknown. In this research, we discovered that LINC00460 is remarkably upregulated in GC tissues compared to the non-tumor tissues. Additionally, LINC00460 served as an independent prognostic marker in GC. Functionally, proliferation of GC cells could be regulated by LINC00460 both in vitro and in vivo. RNA sequencing (RNA-seq) analysis for the whole transcriptome indicated that LINC00460 may serve as a key regulatory factor in the tumorigenesis of GC. What’s more, the biological function of LINC00460 was mediated, to certain extent, by the direct interaction with enhancer of zeste homolog 2 (EZH2) and lysine (K)-specific demethylase 1A (LSD1) proteins. Further analyses indicated that LINC00460 promoted GC proliferation at least partly through the downregulation of tumor suppressor-gene Cyclin G2 (CCNG2), which is mediated by EZH2 and LSD1. In conclusion, our results suggested that LINC00460 acted as an oncogene in GC to inhibit the expression of CCNG2 at least partly by binding with EZH2 and LSD1. Our study could provide additional insights into the development of novel target therapeutic methods for GC.


INTRODUCTION
Gastric cancer (GC) is the fifth most common cancer and the third leading cause of cancer death worldwide. The incidence of GC is relatively high in East Asia (especially in China), and most GC patients are diagnosed at advanced stages when limited efficient treatment methods are available. 1,2 The dysregulation of multiple oncogenes and tumor suppressors has been proved to promote the occurrence of GC through complicated biological processes. 3,4 Given the role of epigenetic dysregulation in tumorigenesis, it is worthwhile to study the comprehensive epigenetic and genetic mechanisms of GC, which could contribute to the exploration of effective biomarkers for diagnosis and therapy of GC, thus improving the clinical outcomes in GC patients. 5,6 The long non-coding RNA (lncRNA) is a sub-category of non-coding RNAs, which is normally composed with more than 200 nucleotides and barely encode any proteins. 7,8 Increasing evidence has shown the critical role of lncRNAs in multiple biological processes in cancer, 9,10 while the regulatory mechanisms for lncRNAs are more complicated than those for small interfering RNAs (siRNAs) and microRNAs (miRNAs). The lncRNAs are closely related to the recurrence, metastasis, and prognosis of various types of cancers due to their regulatory effects on the expression of multiple oncogenes and tumor suppressors via interacting with DNAs, RNAs, and proteins. [11][12][13] Specifically, HOXA11-AS facilitates GC cell proliferation and invasion at the epigenetic level via scaffolding chromatin modification factors PRC2, LSD1, and DNMT1 as a competing endogenous RNA (ceRNA). 14 Additionally, LINC00707 predicts the survival of cancer patients and facilitates GC cell proliferation and migration via interacting with human antigen R (HuR). 15 However, the comprehensive biological functions and specific mechanisms of lncRNAs in GC tumorigenesis and progression need to be further elucidated.
LINC00460 with a length of 913 bp is one of the few well-known lncRNAs and of great significance in various human diseases. [16][17][18][19][20] We previously found that LINC00460 facilitated the proliferation of colorectal cancer cells through modulating the expression levels of KLF2 and CUL4A. 21 In this study, we further explore the role of LINC00460 overexpression in GC cell proliferation and apoptosis as well as the underlying mechanisms. The LINC00460 levels in genome-wide lncRNA expression profiles from the Cancer Genome Atlas (TCGA) database and the specimens from GC patients were determined. The results indicated that LINC00460 significantly increased in human GC tissues relative to non-tumor tissues, the level of which was correlated with disease-free survival (DFS) in GC patients. Functional assay manifested that LINC00460 regulated GC cell proliferation and apoptosis in vitro and in vivo, and RNA sequencing (RNA-seq) analysis showed that LINC00460 gene knockout could primarily affect the genes related to proliferation and apoptosis. From the perspective of mechanism, the functioning of LINC00460 was mediated, at least partially, by binding with EZH2 and LSD1. Furthermore, LINC00460 could also suppress Cyclin G2 (CCNG2) expression by interacting with EZH2 and LSD1. Overall, this study proposed additional insights into the biological role and mechanisms of LINC00460 in GC and further improves the understanding of the regulation of network functions in the malignant progression of GC.

Correlation between Increasing LINC00460 Level in Human GC Tissues and Poor Prognosis
The RNA-seq data of 375 GC tissues and 32 surrounding non-tumor tissues from TCGA were compared. The results indicated that LINC00460 expression significantly increased in tumor tissues relative to non-tumor tissues ( Figures 1A and 1B). The qRT-PCR results of the LINC00460 levels in 80 paired GC tissues and surrounding normal tissues indicated that LINC00460 expression significantly increased in 62 paired tumor tissues ( Figure 1C). Interestingly, LINC00460 was also upregulated in 14 types of cancer tissues analyzed from TCGA, such as head-neck squamous cell carcinoma ( Figure 1D). On the contrary, the level of LINC00460 was lowered in normal gastric tissues from UCSC (https://www.ucsc.edu/) (Figure S1A). Therefore, we speculated that LINC00460 could promote GC tumorigenesis and progression.
The 80 enrolled GC patients were assigned into two groups based on the median level of LINC00460 (higher group; n = 40 and lower group; n = 40). Kaplan-Meier and log-rank tests were employed to assess the relationship between LINC00460 enrichment and DFS. The results revealed that the GC patients with high LINC00460 expression exhibited poorer outcomes compared to the patients with low LINC00460 expression (p = 0.00291) ( Figure 1G). Moreover, univariate and multivariate Cox regression analyses indicated that LINC00460 was an independent prognostic biomarker for GC patients (hazard ratio = 1.671; 95% confidence interval [CI] 1.003-3.976; p = 0.028) ( Figures 1H and 1I).

Regulation of GC Cell In Vitro Proliferation by LINC00460
According to the qRT-PCR results, it was found that, compared to GES1 cells, LINC00460 exhibited higher expression in AGS, BGC823, SGC7901, and MGC803 cells (Figure 2A). Figures 2B and 2C presented the transfection efficacies of si-LINC00460 and LINC00460-overexpressing plasmid (pcDNA-LINC00460). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony-formation analyses illustrated that LINC0460 gene knockout resulted in a marked inhibition of GC cell proliferation, while LINC00460 overexpression promoted the proliferative rates of GC cells ( Figures 2D and 2E; Figures S1B and S1C). To determine the impact of LINC00460 on GC cell cycle and apoptosis, flow cytometry analysis was conducted, and its results demonstrated that the BGC823 and AGS cells with si-LINC00460 transfection exhibited pronounced cell cycle arrest at G0 and G1 phase and a higher apoptotic rate relative to the controls ( Figures 2F and 2G). Similar results were shown in EdU proliferation and TUNEL staining assays ( Figures 2H and 2I).

Promotion of In Vivo GC Tumorigenesis by LINC00460
To evaluate the role of LINC00460 in the in vivo tumorigenesis of GC, BGC823 cells with transfection of sh-LINC00460 and empty vectors were injected into nude mice. After 14 days, the tumors that were harvested from mice in the sh-LINC00460 group were much smaller and lower in weight than those in the control group ( Figures 3A-3C). Meanwhile, LINC00460 levels were lower in the tumors in the sh-LINC00460 group than those in the control group ( Figure 3D). Immunohistochemical (IHC) analysis revealed that the positive expression of proliferation marker Ki-67 in the BGC823 cells transfected with sh-LINC00460 was lower in than the controls ( Figure 3E). These results together indicated that the knockout of LINC00460 may inhibit tumor growth in vivo.

Mechanism Underlying Promotion of Cell Proliferation
To unbiasedly explore the LINC00460-related pathway in GC, RNAseq analysis for BGC823 cells was conducted after silencing LINC00460. As a result, the common set containing 719 mRNAs presented more than 1.5-fold increased abundance, while the knockout of LINC00460 decreased the abundance (less than 1.5-fold) of 554 genes ( Figures 4A and 4B). Kyoto encyclopedia of genes and genomes (KEGG) pathway clustering demonstrated that the most markedly overexpressed biological pathways included cell growth and death, cell motility, and replication and repair ( Figure 4C). Additionally, the dysregulated key genes that were related to the cell cycle and apoptosis pathways included MTPN, IFIT2, IFI44, CDKN1B, CCNG2, ZNF675, KLF3, KLF10, CTGF, WBP11, E2F1, SRF, SULT1A3, TGM2, KLC2, etc. Next, some of these genes were verified using qRT-PCR after the LINC00460 knockout in BGC823 and AGS cells ( Figures 4D and 4E). Subcellular distribution may further provide the clues regarding the molecular mechanism. To explore the underlying mechanism of LINC00460-related regulation on its target genes, we first detected the distribution of LINC00460 transcript in GC cells through subcellular fractionation and RNA-fluorescence in situ hybridization (FISH) analyses. It was found that LINC00460 existed in cytoplasm and nuclei, while the ratio of LINC00460 in nuclei was greater than that in cytoplasm, suggesting that LINC00460 probably played a major regulatory role at the transcriptional level ( Figures 4F and 4G). Given that clear evidence indicated that lncRNAs regulated their target gene expressions via interacting with specific RNA-binding proteins (RBPs), we predicted the correlation between LINC00460 and RBPs at http://pridb. gdcb.iastate.edu/RPISeq/. The results indicated that LINC00460 could bind with EZH2, LSD1, and DNMT1 with random forest (RF) or support vector machine (SVM) scores more than 0.5 ( Figure 4H). To further validate this result, an RNA immunoprecipitation (RIP) assay was performed, and the results demonstrated that LINC00460 only bound with EZH2 and LSD1 ( Figure 4I). Furthermore, an RNA pulldown assay also confirmed that LINC00460 directly bound to EZH2 and LSD1 ( Figure 4J). These findings together suggested that LINC00460 might act as a scaffold binding with EZH2 and LSD1.

Role of the Interactions between LINC00460 and EZH2 or LSD1 in GC Cell Proliferation
According to the Venn analysis, it was shown that seven genes were consistently upregulated, indicating that these genes could be corepressed by LINC00460, EZH2, and LSD1 ( Figure 5A). To confirm whether EZH2 and LSD1 directly bind the promoter regions of these seven genes, we designed three pairs of primers spanning the promoter region of 2,000 bp. A chromatin immunoprecipitation (ChIP) assay demonstrated that LINC00460 knockout inhibited the binding between EZH2 and LSD1 and the promoters of the majority of the co-regulated genes and decreased H3K27 trimethylation and H3K4 demethylation (Figures 5B-5G). Moreover, the data from GEO (GEO: GSE54129) showed that the expression levels of these seven genes markedly decreased in GC tissues compared with normal tissues ( Figure 5H). These findings together indicated that LINC00460 might recruit EZH2 and LSD1 and promote the binding to the above target gene promoters, thus inhibiting transcription.

Regulatory Effect on CCNG2 of LINC00460 and the Potential Involvement in Oncogenic Function
Among the common target genes of LINC00460, EZH2, and LSD1, CCNG2 was of particular interest due to the remarkable fold change in its expression level (Figures 4D and 4E) and significant contribution Colony-formation assay was conducted to assess the effects of LINC00460 knockout or overexpression on cell proliferation. Flow cytometry analysis was conducted to determine cell cycle (F) and apoptosis (G) after LINC00460 knockout. (H) EdU staining was conducted to explore the role of LINC00460 knockout in cell proliferation. (I) TUNEL staining was conducted to measure the cell apoptosis after LINC00460 knockout. Data were presented as mean ± SD of three independent trials. *p < 0.05 and **p < 0.01. to GC tumorigenesis. 22,23 Based on the analysis with the data from GEO (GEO: GSE64951), the expression of CCNG2 was proved to be negatively correlated with LINC00460 ( Figure 6A). In addition, the qRT-PCR performed with 80 pairs of GC and normal tissues also indicated that the mRNA levels of CCNG2 markedly reduced in tumor tissues relative to normal tissues ( Figure 6B). Additionally, a higher level of GC progression was found to be correlated with lower CCNG2 level ( Figure 6C). We also found that 80% of the normal tissues demonstrated the CCNG2-positive signaling, while the majority of the tumor-derived tissues had a lower CCNG2 expression ( Figure 6D). In addition, lower CCNG2 level was also observed in advanced GC stage ( Figure 6E). In total, these data manifested that, conversely to LINC00460, low CCNG2 expression was associated with GC progression.
Subsequently, the potential role of CCNG2 in the LINC00460induced GC proliferation was investigated. Compared with GES1 cells, the CCNG2 expression in BGC823 cells was markedly downregulated ( Figure 7A). Western blotting showed that, compared with the controls, CCNG2 expression was significantly increased in the BGC823 cells that were transfected with CCNG2 vectors, while decreased in those transfected with si-CCNG2 ( Figure 7B). The MTT experiment illustrated that overexpression of CCNG2 suppressed BGC823 cell viability ( Figure 7C), which was also proved to induce BGC823 cell apoptosis ( Figure 7D). The xenograft tumor mouse model was established to uncover the in vivo function of CCNG2 in GC proliferation. As shown in Figures  7E-7G, the mean tumor volume and weight in the CCNG2-overexpressed group were much lower than those in control group. Overall, these data indicated that CCNG2 might act as a tumor suppressor in the malignant progression of GC.
A rescue assay was performed with the BGC823 cells that were co-transfected with LINC00460 and CCNG2 siRNAs to explore the underlying influences of LINC00460/CCNG2 on GC cell growth ( Figure 7H). Colony-formation and MTT assays indicated that the co-silence of LINC00460 and CCNG2 could partially reverse the proliferation change induced by LINC00460 downregulation in BGC823 cells (Figures 7I and  7J). Finally, LINC00460 knockout was found to significantly change the manifestations of the key genes related to tumorigenesis in BGC823 and AGS cells, including CDK2, WBP11, E2F1, and p21 ( Figure 7K).

DISCUSSION
Many GC patients were diagnosed at the advanced stage of GC due to the lack of early-detection techniques. 24 More seriously, the most critical biological features of GC are uncontrolled cell proliferation and apoptosis, which are the major reasons for death. 15,25 GC carcinogenesis was regarded as a multistage process involving genetic and epigenetic alterations. 4,6 Currently, lncRNAs have been considered as active biological molecules rather than "transcriptional noise." In human cancers, hundreds of lncRNAs have been found by RNA-seq and were recorded in databases such as TCGA. 26 They were proved to drive carcinogenesis via regulating various cellular processes, including the control of gene expression and protein translation. 9 Moreover, lots of lncRNAs were also reported to participate in the tumorigenesis and progression of GC, including HOXC-AS3, LINC01234, GMAN, and FOXD2-AS1. [27][28][29][30] The key finding of this study was the overexpression and prognostic significance of LINC00460 in GC tissues according to the TCGA analysis of clinical specimens. Kaplan-Meier analysis illustrated that the higher expression level of LINC00460 was correlated with poor survival outcomes in GC patients. Multivariate analysis showed that LINC00460 served as an independent prognostic biomarker of DFS in GC patients.
Moreover, the pathological and tumorigenic roles of LINC00460 in GC were also revealed, that LINC00460 promoted the proliferation but inhibited the apoptosis of GC cells. Consistent with our findings, Zhang et al. 31 also found that LINC00460 enhanced GC cell proliferation and invasion through activating the Wnt/b-catenin signaling pathway. Therefore, our study clearly indicated that LINC00460 could sever as a potential prognostic and diagnostic biomarker of GC.
Increasing evidence indicated that lncRNA could regulate cancer cell phenotypes by influencing the expression of target genes through  various mechanisms, such as chromatin interaction. 32 It was reported that LINC00460 served as a ceRNA that sponges miRNAs to regulate gene expression. 18,33 In this study, we found that LINC00460 was primarily located at the nuclei of GC cells, indicating that it may impose impacts at transcriptional level. RIP and RNA pull-down assays demonstrated the binding between LINC00460 and EZH2 and LSD1, which confirmed our findings. To unbiasedly explore the pathways that were related to the effects of LINC00460 in tumorigenesis of GC, RNA-seq analysis was conducted, and several protein-coding genes were found to be regulated by LINC00460, EZH2, and LSD1. Thus, LINC00460 was believed to recruit EZH2 and LSD1 to the pro-moter regions of target genes and suppress their transcriptions through mediating histone H3 at lysine 27 (H3K27me3) trimethylation and H3K4me2 demethylation.
CCNG2 is an atypical cyclin, which could negatively regulate the cell cycle, and its expression is observed to be downregulated in multiple human cancers. 22,34,35 Recently, it has been proved that certain microRNAs could promote tumor progression by suppressing CCNG2 expression. [36][37][38] In our study, CCNG2 was found to be markedly upregulated after LINC00460 knockout, and the overexpression of CCNG2 could induce apoptosis and suppress proliferation of GC cells. Moreover, the results also indicated that the inhibitory effect on CCNG2 expression of LINC00460 could be achieved via recruiting EZH2 and LSD1 to the promoter regions of target genes by the LINC00460-mediated H3K27 methylation and H3K4 demethylation.
In summary, our study illustrated that lncRNA LINC00460 expression was upregulated in GC tissues and cells, and its high level could be associated with poor prognosis in GC patients, which made it a potential negative prognostic factor for GC. As an oncogenic effector, LINC00460 epigenetically silenced CCNG2 (IFIT2, IFI44, KLF2, and P21) expressions by binding with EZH2 and LSD1, thus influencing the proliferation and apoptosis of GC cells (Figure 8). Our findings provided additional evidence that LINC00460 could be a possible therapeutic target that is beneficial to broaden current understanding of treatment strategy for GC. However, further studies are still needed to fully explore the possible binding partners of LINC00460 as well as upstream factors.

Analysis for Gene Expression Profiling
In this study, GSE54129 and GSE64951 datasets were employed, and the GC gene expression data in the GEO dataset were obtained from TCGA. The BAM and normalized probe-level intensity files were obtained from TCGA and GEO databases, respectively. The probe sequences were acquired from GEO and microarray manufacturers. The bowtie was employed to re-annotate probes based on the GENCODE release 19 annotation for lncRNAs. Figure 7. Ectopic CCNG2 Expression Suppresses GC Cell Proliferation, and CCNG2 Is a Good Target for LINC00460 (A) qRT-PCR analysis for the CCNG2 mRNA expression in GES1 and BGC823 cells. (B) Western blotting was conducted to detect CCNG2 expression after CCNG2 knockout or overexpression in BGC823 cells. (C) MTT assay was used to determine the viability of the BGC823 cells transfected with CCNG2 and empty vectors. (D) Cell apoptosis was determined using flow cytometry analysis after the CCNG2 overexpression in BGC823 cells. (E) BGC823 cells were injected into nude mice after the transfection of empty and CCNG2 vector to assess the effects of CCNG2 overexpression on GC tumorigenesis. (F) Tumor volumes were measured every 3 days. (G) Tumor weights in two groups were shown. (H) Western blotting was conducted to determine the CCNG2 expression in the BGC823 cells with the co-transfection of si-LINC00460, si-CCNG2, and si-NC. Colony-formation (I) and MTT (J) assays were conducted to measure the viability of the BGC823 cells with co-transfection of LINC00460 and CCNG2 siRNAs. (K) Protein levels of the selected genes in the GC cells with LINC00460 knockout were measured by western blotting. Data were presented as mean ± SD of three independent trials. *p < 0.05 and **p < 0.01.

GC patients receiving surgery at Zhongshan Hospital, Xiamen
University between January 2007 and December 2016 were included in our study. The diagnosis was made and verified by an experienced pathologist. DFS was defined as the interval from the onset of surgery to the time point of recurrence. If no tumor recurrence was observed within the follow-up period, patients were censored on the date of death or the last day of the follow-up period. The tissues acquired during surgeries were frozen in liquid nitrogen and stored at À80 C for subsequent experiments. None of the patients received preoperative radiotherapy or chemotherapy. Informed consents were obtained from all participators, and all the experimental procedures were approved by the Ethics Committee of Xiamen University.

Cell Culture
Four human GC cell lines (BGC823, AGS, SGC7901, and MGC803) and a gastric epithelium cell line (GES1) were acquired from the Cell Bank of the Chinese Academy of Sciences. Cells were cultured in RPMI 1640 or DMEM (Gibco) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin in 5% CO 2 at 37 C.

qRT-PCR
Total RNA was first extracted from cells and tissue samples using TRIzol reagent (Invitrogen). Then, 1 mg RNA was reversely transcribed using PrimerScript RT master mix (Takara). Finally, qRT-PCR was carried out as previously reported. 21 All primer sequences for qRT-PCR were summarized in Table S1.

Cell Transfection
GC cells were inoculated into 6-well plates and transfected with specific (100 nM) and negative control (NC) siRNAs (100 nM) by Lip- ofectamine 2000 (Invitrogen). 48 h later, these cells were collected for subsequent qRT-PCR and western blotting analyses. All primer and siRNA sequences were presented in Table S1. DNA Midiprep or Midiprep kits (QIAGEN) were used to generate the transfected plasmid vectors (pcDNA-LINC00460, sh-LINC00460, pcDNA-CCNG2, and empty vector).

Cell Proliferation Assay
MTT, colony formation, and ethynyldeoxyuridine (EdU) assays were conducted to measure cell proliferation. The MTT assay was first conducted based on the instructions of kit (Sigma). Then, cells were inoculated into 6-well plates and incubated with 10% FBS-supplemented medium for 2 weeks to perform the colonyformation assay. The assay was carried out in triplicate. Colonies were fixed with methanol and stained with 0.1% crystal violet (Sigma-Aldrich) for 15 min to count the stained colony numbers, thereby measuring the clonality. EdU assay was conducted based on the instructions of 5-ethynyl-2-deoxyuridine labeling and detection kit (Ribobio). In this assay, nuclei were stained by DAPI. Five fields were randomly selected from each well. The EdU-positive cells were observed and counted under a fluorescent microscopy.

Flow Cytometry Analysis
Cells were stained by fluorescein isothiocyanate (FITC)-annexin V and propidium iodide (PI) to detect cell apoptosis in a flow cytometer (BD Biosciences) with Cell Quest software. In this software, cells could be categorized into living cells, dead cells, and early and late apoptotic cells. Finally, the apoptotic rate was calculated. For the detection of cell cycle, cells were stained with PI based on the instructions of the CycleTEST PLUS DNA reagent kit (BD Biosciences) and analyzed in the flow cytometer to calculate the percentage of cells at G0-G1, S, and G2-M phases.

TUNEL Assay
A TUNEL assay was conducted based on the instructions of an apoptosis detection kit (KeyGEN BioTECH, China). Under a fluorescent microscopy, the TUNEL-positive cells in five random fields per well were observed and counted to calculate the percentages of TUNEL-positive cells.

Xenograft Mouse Model
Athymic mice were provided by the Animal Center of the Chinese Academy of Science and housed in a laminar flow cabinet without specific pathogen. For the in vivo proliferation assay, BGC823 cells were transfected with small hairpin RNA (shRNA) and empty www.moleculartherapy.org vectors, respectively. Then the transfected cells were administrated into nude mice. The tumor volumes and weights were determined every 2 days. Tumor volume was measured as length Â width 2 Â 0.5. After 2 weeks of injections, mice were killed to harvest tumors. The tumor samples were sliced and stained by using H&E and IHC. 21 The animal experimental procedures were approved by the Animal Care and Use Committee of Xiamen University.

IHC Analysis
To quantify the CCNG2 expression in the tumor tissues that were harvested from mice, we assessed the intensity and extent of immune reactivity. The intensity was scored in accordance with following standards: negative, 0; weak, 1; moderate, 2; and strong, 3. The extent was also scored from 0 to 3 based on the percentage of the CCNG2positive cells per field under fluorescent microscopy (<25%, 0; 25%-50%, 1; 50%-75%, 2; 75%-100%, 3). Finally, the scores of intensity and extent were multiplied to obtain the total score ranging from 0 to 9. If there is a disagreement (score discrepancy > 1), it is necessary to consult with experts and to re-examine the slides, thereby reaching a consensus.

Subcellular Fractionation and Fluorescence In Situ Hybridization
The nuclear and cytosolic fractions were separated based on the instructions of the PARIS kit (Life Technologies). Then, reverse transcription reaction and RT-PCR were carried out by using the RNA from each fraction. All primer sequences were presented in Table  S1. For the FISH assay, 4% formaldehyde was used to fix GC tissues and cells for 15 min. After washing with PBS, the samples were treated with 1% pepsin and then dehydrated in ethanol of different concentrations. Next, samples were dried at room temperature and treated with 40 nM FISH probe at 80 C for 2 min. The hybridization was conducted at 55 C for 2 h, and then the slides were washed and dehydrated. Finally, the dried slides were stained by using DAPI. The RNA FISH probe was synthesized by Bogu (Shanghai, China), and its sequences were shown in Table S1.

RIP Assay
The RIP assay was conducted based on the instructions of the Magna RIP RNA-binding protein immunoprecipitation kit (Millipore, USA). Anti-EZH2, anti-LSD1, and anti-DNMT1 antibodies for RIP were provided by Millipore. A RT-PCR assay was conducted to measure the co-precipitated RNAs.

RNA Pull-Down Assay
The RNA pull-down assay was performed according to the instructions of the Magnetic RNA-protein pull-down kit (Pierce Biotechnology, USA). The RBPs were detected using western blotting.

ChIP Assay
The ChIP assay was carried out based on the instructions of a chromatin immunoprecipitation kit (Millipore). Immunoprecipitations with anti-LSD1, anti-H3K4me2, anti-EZH2, anti-H3K27me3, or anti-immunoglobulin G (IgG) as NC were measured. Primers were designed according to the sequences of target gene promoters to conduct qRT-PCR, and their sequences were listed in Table S2.

Statistical Analyses
Continuous variables were presented in the form of mean ± SD. Twotailed Student's t and Kruskal-Wallis tests were conducted to assess the differences between two groups. Kaplan-Meier curves were drawn, and a log rank test was adopted to assess the DFS. A univariate Cox proportional hazard model was used to evaluate survival data. Significant variables were used to construct the multivariate Cox regression model. Prism 5 software (GraphPad) was employed to calculate the Pearson's correlation coefficient. All the analyses were performed with SPSS 20.0 software. A p value less than 0.05 was considered as statistical significance.