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LncRNA HRCEG, regulated by HDAC1, inhibits cells proliferation and epithelial-mesenchymal-transition in gastric cancer
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LncRNA HRCEG, Regulated by HDAC1, Inhibits Cells Proliferation and Epithelial-Mesenchymal-Transition in Gastric Cancer Shuheng Wu , Erzhong Wu , Dongpeng Wang , Yiwei Niu , Haiyan Yue , Dongdong Zhang , Jianjun Luo , Runsheng Chen PII: DOI: Reference:
S2210-7762(19)30558-7 https://doi.org/10.1016/j.cancergen.2019.12.007 CGEN 8588
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Cancer Genetics
Received date: Revised date: Accepted date:
13 October 2019 25 December 2019 25 December 2019
Please cite this article as: Shuheng Wu , Erzhong Wu , Dongpeng Wang , Yiwei Niu , Haiyan Yue , Dongdong Zhang , Jianjun Luo , Runsheng Chen , LncRNA HRCEG, Regulated by HDAC1, Inhibits Cells Proliferation and Epithelial-Mesenchymal-Transition in Gastric Cancer, Cancer Genetics (2020), doi: https://doi.org/10.1016/j.cancergen.2019.12.007
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Highlights
One LncRNA HRCEG inhibits cell proliferation and EMT process in gastric cancer.
HRCEG was significantly connected with gastric cancer related pathways and were also highly involved in the EMT processes.
The expression of HRCEG is regulated by HDAC1.
LncRNA HRCEG, Regulated by HDAC1, Inhibits Cells Proliferation and Epithelial-Mesenchymal-Transition in Gastric Cancer Shuheng Wu1,2+, Erzhong Wu1,2+, Dongpeng Wang1,2+, Yiwei Niu1,2, Haiyan Yue1, Dongdong Zhang1, Jianjun Luo1,2*, and Runsheng Chen1,2,3* 1 Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China 2 University of Chinese Academy of Sciences, Beijing, 100049, China 3 Guangdong Geneway Decoding Bio-Tech Co. Ltd, Foshan, 528316, China +
The authors wish it to be known that, in their opinion, the first three aothors should be regarded as Joint First Authors. The authors declare no competing financial interest. * Corresponding author at: Institute of Biophysics, Chinese Academy of Sciences, No. 15, Datun Road, Chaoyang District, Beijing 100101, China. Email addresses: [email protected] (J.Luo), [email protected] (R.Chen) Abstract Recently, a number of long noncoding RNAs (lncRNAs) have been reported to play significant roles in human tumorigenesis. However, only few gastric cancer related lncRNAs have been well characterized. Here, we identified one lncRNA HRCEG, whose expression was decreased in the gastric cancer tissues compared with adjacent normal tissues. Overexpression of HRCEG significantly promoted cell apoptosis and inhibited cell proliferation. Importantly, we demonstrated that HRCEG levels inversely correlated with EMT process and HRCEG was regulated by the histone deacetylase 1 (HDAC1) in gastric cancer. These findings suggest that HRCEG might be regulated by HDAC1 to inhibit gastric cancer progress and metastatic capability via EMT pathway. 1. Introduction Gastric cancer (cardia and noncardia gastric cancer combined) is an important cancer worldwide and is the fifth most frequently diagnosed cancer and the third leading cause of cancer death (Bray et al., 2018). Different from other gastrointestinal cancer, the incidence rate of gastric cancer is especially high in Eastern Asia (Rahman et al., 2014). Given the significant development of surgical technology and endoscopy, the 5-year mortality has been decreasing particularly for the early gastric cancer. However, for advanced gastric cancer, the survival rate is still less than 15% once metastasizes (Kang et al., 2014). In recent years, many molecular mechanisms of gastric cancer have been investigated, but the mechanism of gastric cancer progression such as proliferation, growth, migration, invasion and apoptosis is still limited and incomplete. Numerous studies have demonstrated that long non-coding RNAs may play important roles in gastric cancer progression (Song et al., 2017; Xu et al., 2017; Zong et al., 2019). Long non-coding RNAs (lncRNAs) are a class of ncRNAs longer than 200 nucleotides and
lack of protein-coding ability (Chen et al., 2014; Rinn and Chang, 2012). More recently, more than 172,216 lncRNA transcripts for human have been collected in our latest version of NONCODE database (Fang et al., 2018). LncRNAs act as activators, decoys, guides, or scaffolds for their interacting proteins, DNA and RNA (Yao et al., 2019). Increasing evidence has suggested that lncRNAs could play essential roles in almost all the biological processes, including stem cell maintenance (Wang et al., 2015; Wang et al., 2019), cell proliferation (Sun et al., 2017), cell apoptosis (Xu et al., 2015), migration (Hao et al., 2017), and epithelial-to-mesenchymal transition (EMT) in several cancers (Li et al., 2018). In the case of gastric cancer, however, there is still little knowledge about gastric cancer-specific lncRNAs, and the specific functional roles of these molecules are still unclear. Although several lncRNAs have been found to participate in gastric cancer to date, such as PVT1, MALAT1, DANCR, NEAT1, LINC00673 (Sawaki et al., 2018), we believe that there are still many unexplored differentially expressed lncRNAs in gastric cancer, especially their expression pattern and underlying regulation. In this study, we functionally characterized one lncRNA HRCEG (HDAC1 regulated RNA affecting Cell proliferation and EMT transition in Gastric cancer), a 3.8 kb antisense RNA of solute carrier family 25 member 25 (SLC25A25) gene. We found that expression of HRCEG was decreased in gastric cancer and overexpression of HRCEG inhibited gastric cancer cells proliferation and EMT process. The low expression of HRCEG in gastric cancer was regulated by HDAC1. These results suggest that further studies are needed to detail the functional mechanism of HRCEG in GC and HRCEG could be a new diagnostic biomarker and therapeutic target. 2. Materials and Methods 2.1. Tissue samples In this study, 18 pairs of gastric cancer samples were collected from Chinese PLA 301 Hospital. All samples contained pairs of gastric cancer tumor and adjacent normal tissue for qRT-PCR validation. This study was informed and obtained consent from each patient before their participation. All tissue samples were collected by surgical operation, fla sh freezing in liquid nitrogen and transferred to -80℃ freezer for storage. 2.2. Cell culture and Cell line transfection Human gastric cancer cell line, MKN28 and BGC823 were purchased from American Type Culture Collection (ATCC) and cultured with the suggestions in ATCC. Human gastric cancer cell line, MKN28 and BGC823 were cultured with DMEM medium(Life Technologies, 11995-065), supplemented with 10% fetal bovine serum(Life Technologies, 16000-044), 1% penicillin, and 1% streptomycin(Life Technologies, 10378-016 ) in a humidified 5% CO2 atmosphere at 37 ℃. For pcDNA3.1 and p-Enter plasmid transfection, cells were transfected with plasmids using Lipofectamine 2000 (Invitrogen, 11668-019) as suggested approaches. 2.3. Bioinformatics Analyses LncRNAs and mRNAs were identified from the gene expression data GSE15459 and GSE79973. Paired-t-test was used to obtain the differentially expressed genes using a Limma
package in R. The GseaPreRanked tool in GSEA v2.0 was used to perform GSEA of HRCEG. 2.4. qRT-PCR Total RNA was extracted by TRIzol reagent (Invitrogen, 15596-026) according to the manufacturer's instructions, and RNA was subjected to DNaseI (Invitrogen, AM2222) treatment. qRT-PCR was performed using the TransScript II Green One-Step qRT-PCR SuperMix kit (TransGen Biotech, AQ311-01) with 100 ng RNA as template in a 20 μl reaction volume on the Rotor-Gene® Q real-time cycler (Qiagen). The results were normalized with GAPDH, and the RNA relative expression was calculated by the ΔΔCt method. The primer sequences are listed in Supplemental Table 1. 2.5. shRNA-mediated interference and lentivirus packaging. shRNAs against HRCEG were designed using online software (https://rnaidesigner.thermofisher.com/rnaiexpress/design.do). Two shRNAs were designed, and a non-targeting, scrambled silencing RNA (scr) was used as a negative control. shRNAs targeting HRCEG and the scr hairpins were cloned into pSicoR-puro lentiviral vector. Virus packaging was carried out in 293T (ADCC) cell line after co-transfection of recombinant lentivirus expression plasmids for shHRCEG together with the packaging plasmids (PMD2G, psPAX2, Addgene) using Lipofectamine 2000 (Invitrogen, 11668-019). The virus was harvested at 48h after transfection, and infected BGC823 cell line for 24h. Then fresh medium with puromycin (2 μg/ml) was added to select for positive cells for additional 3 days incubation. The RNAi efficiency of HRCEG was checked by qRT-PCR analysis. 2.6. RNA interference siRNAs used to knock down HDAC1 mRNAs were designed online (https://rnaidesigner.thermofisher.com/rnaiexpress/design.do) and synthesized by the GenePharma company. A nonspecific siRNA used as the negative control (NC) was also purchased from the GenePharma Company. MKN28 and BGC823 cells were seeded in 6-well plates at a desired concentration for 12h and transfected with siRNA oligonucleotides with Lipofectamine 2000 according to the manufacturer's instructions. 2.7. Cell migration and invasion assay For knockdown and overexpression of HRCEG, BGC823 cells were infected with shHRCEG virus and transfected with pcDNA3.1-HRCEG plasmids, trypsinized and counted after 48 h. A total of 60,000 cells (for the migration assay) or 10,000 cells (for the invasion assay) in 100 μl serum-free medium were seeded in the upper chamber of Transwell plates (Corning, 3422) with (for invasion) or without (for migration) BD Matrigel matrix (BD, 356230) coating. 600 μl medium containing 20% FBS was added into the lower chambers as the chemotactic factor. After 10h cell culture at 37℃ and 5% CO2, the non-migrating or non-invading cells in upper chamber were removed, and the migrating or invading cells in lower chamber were stained with 0.1% crystal violet for 20min, then washed with water, air dried and photographed using Nikon microscope imaging systems. The migrating and invading cells were counted and averaged from images of six random fields (magnification was 20× or 40×) for differential analysis.
2.8. Cell proliferation and apoptosis Cell proliferation ability was examined using a Cell Proliferation Reagent Kit I (MTS; Promega, G3580). MKN28 and BGC823 cells were harvested and resuspended in PBS. Then the cells were stained with Dead Cell Apoptosis Kit with Annexin V Alexa Fluor™ 488 & Propidium Iodide (PI) (Invitrogen, V13245) as instructions and analyzed by Flow Cytometry (BD FACS Calibur, USA). 2.9. Subcellular fractionation The nuclear and cytoplasmic fractions of MKN28 and BGC823 cells were isolated with the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo, 78833). The nuclear and cytoplasmic location of HRCEG was assessed by qRT-PCR on RNA isolated from the subcellular fractions. GAPDH and ACTB were served as the cytosolic controls while U1 and MALAT1 was used as the nuclear control. 2.10. Western Blot Western blotting was performed as standard protocols. And the primary antibodies used in this study were obtained as follows: ACTB (Proteintech, 66009-1-Ig ), Snail (CST,C1503), Vimentin (Invitrogen, MA1-10459), E-cadherin (CST, 144725), Cleaved CASP3 (ABGENT, Asp175). 2.11. RNA fluorescence in situ hybridization In vitro transcription of DIG-labeled 200bp antisense HRCEG RNA probe was conducted using T7 RNA polymerases (Roche) and DIG RNA Labeling Mix (Roche). Probe template was amplified with ExPfu PCR StarMix (Genstar, A063-01). Synthesized probes were purified using RNeasy® mini kit (Qiagen). RNA FISH was performed as described in (http://wmc.rodentia.com/docs/Big_In_Situ.html) with minor modifications. Briefly, after fixed with 4% formaldehyde for 10min, cells cultured on coverslips were permeabilized with 1x PBS/0.5% Triton X-100 followed by acetylation and pre-hybridization. Hybridization was performed with probes at a concentration of 1µg/ml at 65°C overnight. For detection of dig labelled probes, the coverslips were incubated with mouse monoclonal Anti-Digoxigenin antibody (Abcam) (1:200) for 1h at room temperature, and signals were visualized by TSA Fluorescein System (PerkinElmer, 744001). The coverslips were then washed, mounted with SlowFade Gold Antifade Reagent (Invitrogen, p36930) and observed with an Olympus FV1200 confocal microscope. Primer sequences were shown in Supplemental Table 1. 2.12. 5′ and 3′ RACE. 5′ and 3′ RACE of HRCEG were performed using the SMARTer® RACE 5’/3’ Kit (Takara, 634858) as recommended by the manufacturer. RACE PCR products were obtained using ExPfu PCR StarMix (Genstar, A063-01), the appropriate gene-specific primers are summarized in Supplemental Table 1. RACE PCR products were separated on a 1% agarose gel. Gel products were extracted with the Gel and PCR Clean-Up System (Promega, A9282), cloned into the pGEM-T Vector Systems I (Promega, A3600), and sequenced bi-directionally using the M13 forward and reverse primers by Sanger sequencing at Sangon Biotech. At least five colonies were
sequenced for every RACE PCR product that was gel purified. 2.13. Chromatin immunoprecipitation (ChIP) and quantitative analysis HDAC1 antibody (abcam, ab7028) was used to perform the ChIP assay in MKN28 and BGC823 cells. ChIP assays were carried out as described previously (Nelson et al., 2006). Briefly, MKN28 and BGC823 cells were cross-linked by 1% formaldehyde for 10min and the reaction was quenched with 250 mM glycine. After washing with cold DEPC-PBS, fixed MKN28 and BGC823 cells were pelleted and resuspended in 1ml sonication buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA). Cell suspension was sonicated in ice bath to generate chromatin fragments of ~500bp in length. Chromatin lysate was cleared by centrifugation and diluted to 10 folds with dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, pH8.0). 1% volume of the sample was taken as input DNA and 4 µg antibodies against HDAC1 were added to the samples and rotated at 4 °C overnight. After hybridization, 50 µl of Protein A/G beads (Thermo Fisher, 88802) was added to each hybridization reaction. Beads were captured by DynaMag-15 magnetic strip and washed with 1 ml low salt buffer (0.1% SDS, 20 mM Tris-HCl, pH 8.0, 1% Triton X-100, 150 mM NaCl) once, high salt buffer (0.1% SDS, 20 mM Tris-HCl, pH 8.0, 1% Triton X-100, 500mM NaCl) once, LiCl buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.25 mM LiCl, 0.1% NP-40, 1% deoxycholate sodium) once and TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) twice. 100 µl Elution buffer (0.1 M NaHCO3, 1% SDS and proteinase K) and 8 µl 5M NaCl was used to reverse cross-linking of DNA–protein complexes at 65 °C for 4–5h. DNA was purified by phenol-chloroform extraction and subjected to qPCR. All primers used in this study are listed in Supplemental Table 1. 2.14. Statistical Analysis All statistical analyses were performed using Graphpad Prism 7.0 statistical software (California, US). Experimental data are shown as the means ± SD, all experiments were conducted at least three times. Significance was determined using the Student's t-test or two-way ANOVA: *, p<0.05; **, p<0.01 and ***, p<0.001. 2.15. Data Availability All data generated during this study are available from the corresponding author on reasonable request.
3. Results 3.1. HRCEG significantly under-expressed in gastric cancer Several lncRNAs are found performing very important functions in alimentary canal cancers (Chen et al., 2018; Guo et al., 2018; Sun et al., 2017; Yan et al., 2017), especially in colorectal cancer and gastric cancer. In a course of studying the transcriptional profiling analysis and functional prediction of long noncoding RNAs in cancer (Yuan et al., 2016) as well as transcriptional signature analyses for gastric cancer (data not shown), we also noticed one gastric cancer specific differentially expressed unknown lncRNA, herein termed as HRCEG. To further investigate the roles of HRCEG in gastric cancer, we first analyzed two additional microarray data from NCBI GEO database (i.e. GSE15459 and GSE79973). We found that HRCEG significantly decrease in gastric cancer compared with paired-adjacent non-tumor (Figure 1A). Furthermore, we evaluated expression pattern of HRCEG using 18 pairs of gastric cancer tissues and adjacent normal tissues from patient samples (Figure 1B). To characterize HRCEG in detail, we used RACE assay to identify the full sequence of HRCEG in MKN28 cell line based on the sequence archived in RefSeq database of NCBI. Sequence with 3.8kb in length was successfully amplified and aligned (Figure 1C), which is a little longer than archived in the RefSeq database. Full length PCR amplification with primers designed according to RACE produced sequence also yielded a product with size similar to the RACE result of HRCEG (Figure 1D). Furthermore, as localization of lncRNA reflects typical function categories of RNA, next, we examined the subcellular localization of HRCEG in MKN28 cell line by nuclear-cytoplasmic fractionation (Figure 1E) and RNA fluorescence in situ hybridization (FISH) (Figure 1F), and data clearly indicated that the majority of HRCEG transcript resides in nucleus. These results indicated that we identified one nuclear localized lncRNA HRCEG, whose expression was potentially repressed in the gastric cancer tissues compared to nearby normal tissues. 3.2. HRCEG regulate gastric cancer cell proliferation and apoptosis To further investigate the physiological roles of HRCEG in gastric cancer, we performed gain-of function studies in gastric cancer cell lines MKN28 and BGC823. As shown in Figure 2A, RT-qPCR show that the pCDNA3.1-HRCEG vector could significantly increase the expression of HRCEG. Then, we measured cell viability in both HRCEG high expressed BGC-823 and MKN28 cell lines by MTS assay. Compared with control cells transfected with empty vector, overexpression of HRCEG inhibited cell proliferation (Figures 2B, C). Additionally, we also examined the effect of HRCEG on apoptosis by Annexin-V and FACS analysis, and found that cell apoptosis significantly increased upon the up-regulation of HRCEG (Figures 2D, E). Consistently, we also observed that overexpression of HRCEG resulted in more Caspase3 cleavage compared to control cells (Figure 2F). These results indicated that HRCEG might inhibit gastric cancer progress by regulating cell proliferation and apoptosis. To assess the molecular pathways that HRCEG may be involved in, we performed Gene Set Enrichment Analysis (GSEA) of HRCEG associated gene sets by Pearson correlation coefficients between expression profiles of mRNA and HRCEG (GEO15459). The results revealed that the
gene sets enriched in gastric cancer progress, EMT, cell apoptosis, cell cycle and cancer related pathways (Figures 3A-F). The correlation between HRCEG and the clinical features of cancer patients also showed that HRCEG expression levels was lower in invasive subtype (P = 0.001) (Figure 3G). Kaplan-Meier survival analysis supported that lower HRCEG expression correlated with worse overall survival, indicating that decreased expression of HRCEG is correlated with gastric cancer progression and poor prognosis (Figure 3H). 3.3. HRCEG suppresses EMT process in gastric cancer cells As our results showed that HRCEG may function as a tumor suppressor in gastric cancer, we next investigated whether HRCEG was involved in the EMT process. The GSEA of the dataset GSE15459 indicated a high correlation between HRCEG and EMT process (Figure 3B). To study this further, we performed the transwell assays to measure cell metastatic capability. As shown in Figures 4A, RT-qPCR show that designed shRNAs could significantly decreased the expression of HRCEG. HRCEG knock-down by specific shRNA treatment increased the invasive capacity of BGC823 cells, whereas, overexpression of HRCEG inhibited the invasion of BGC823 cells (Figures 4B, C). In the migration assay, we also found that down-regulation of HRCEG significantly reduced the migration ability of BGC823 cells and up-regulation of HRCEG promoted BGC823 cell migration. Bioinformatics analysis showed that HRCEG had a significantly negative correlation with EMT related genes in GSE15459 dataset (Figure 4F). Then, we detected the expression level changes of the EMT markers following HRCEG knockdown and overexpression by Western blotting. As shown in Figure 4G, HRCEG overexpression remarkably increased the expression of E-cadherin but decreased expressions of mesenchymal-related genes, including Snail and Vimentin. Consistently, we also detected that lower expression of E-cadherin and higher expression of Snail and Vimentin upon down-regulation of HRCEG (Figure 4H). Taken together, these data show that HRCEG influence gastric cancer progression by affecting the EMT processes. 3.4. HDAC1 suppressed HRCEG transcription in gastric cancer cells Previous reports demonstrated that epigenetic modification could regulate lncRNAs transcription in cancer cells such as methylation and acetylation (Dong et al., 2018; Rokavec et al., 2017; Zhang et al., 2017). It is known that histone deacetylase 1 (HDAC1) could erase the acetylation status of core histone, regulate genes transcription and play important regulatory roles in cancer cells (Weichert et al., 2008). HDAC1 expression has been reported to be up-regulated in gastric cancer (Mutze et al., 2010), and we found HRCEG expression level was associated with AJCC pathology in gastric cancer using the canSAR 1 (Figure 5A). Genes correlated with HRCEG shown in GSEA analysis were also enriched for HDAC1 targets, which suggests the possibility that HRCEG is regulated by HDAC1 in gastric cancer cells. Using ChIP-qPCR assays, we found that HDAC1 could enrich to the HRCEG gene region (Figure 5B). To investigate the relationship between HRCEG and HDAC1, we knockdown and up-regulate HDAC1 in gastric cancer cells (Figures 5D, E). The expression of HRCEG increased upon knockdown of HDAC1 and decreased upon overexpression of HDAC1 (Figures 5D, E). In conclusion, these data confirmed that HRCEG is regulated by HDAC1 and may explain the reason of low expression of HRCEG in gastric cancer.
4. Discussion LncRNAs are emerging components that are recognized to play critical roles in cancer development and progression. Recently, some lncRNAs have been found to participate in gastric cancer tumorigenesis. LncRNAs function by impacting proliferation, metastasis, migration, and epithelial-to-mesenchymal transition (EMT). Here, we demonstrated that lncRNA HRCEG, which is mainly localized in the nucleus and expression level significantly decreased in gastric cancer tissues compared with adjacent normal tissues and cell lines, including BGC823 and MKN28. Thus, expression of HRCEG may be required to repress gastric cancer tumorigenesis and progression. It has been shown that lncRNAs play key roles in regulation of the malignant phenotypes of cancer cells. In this study, we found that overexpression of HRCEG inhibited gastric cancer cell proliferation, and induced cell apoptosis. Our findings suggest that HRCEG may function as a tumor suppressor lncRNA in gastric cancer and potentially be considered as a novel prognostic indicator for gastric cancer. We further determined the expression levels of apoptosis-related proteins. Results show that the inhibition on HRCEG promoted the accumulation of apoptotic protein caspase-3. Caspase-3 is a frequently activated death protease, catalyzing the specific cleavage of many key cellular proteins (Porter and Janicke, 1999). Activation of pro-caspase-3 is a central event in the execution phase of apoptosis and appears to serve as the convergence point of different apoptotic signaling pathways. Increasing evidence has demonstrated a potential role of lncRNAs in gastric cancer metastasis by influence the EMT process. Our GSEA data illustrated that HRCEG was significantly connected with gastric cancer related pathways and were also highly involved in the EMT processes. Furthermore, our cell invasion and migration assays revealed that HRCEG indeed play regulatory roles on gastric cancer metastasis. The feature of EMT occurrence is that the epithelial marker E-cadherin is down-regulated and mesenchymal marker, like Snail and Vimentin, is up-regulated. To demonstrate whether HRCEG affected EMT markers, we detected the molecular marker levels of EMT and showed the inhibition of HRCEG on EMT. Histone Deacetylase 1 (HDAC1) is tightly correlated with gastric cancer, and its high expression has been shown to correlate with advanced stage, uncontrolled tumor cell proliferation and poor prognosis in gastric cancer, which implies that HDAC1 has a critical role in carcinogenesis. HDAC1 was recently identified as an epigenetic regulator through modulation of FENDRR transcriptional activation, and HDAC1 contributed to the decreased expression of FENDRR in gastric cancer cells. Besides, FENDRR suppresses gastric cancer cell metastasis in vitro and in vivo, and a negative correlation between fibronectin (FN)1 and FENDRR expression was reported. Thus, we further determined whether HDAC1 is a regulator of HRCEG disorder expression in gastric cancer cells. Interestingly, our results confirmed that HDAC1 down-regulates HRCEG expression in gastric cancer, and decreased HRCEG expression induces Snail and Vimentin expression. Our findings revealed that HRCEG inhibits cell proliferation and EMT process in gastric
cancer. The expression of HRCEG is regulated by HDAC1. More works are needed to characterize the underlying mechanism how HRCEG serves as a regulatory factor in the gastric cancer. Funding This work was supported by grants from the National Key Research and Development Program of the Ministry of Science and Technology of China (2017YFC0907503 and 2018YFA0106901), as well as grant from the National Natural Science Foundation of China (31520103905). Conflict of interest The authors declare that they have no competing interests. Acknowledgements We are very grateful to Dr. Hangcheng Huang and Ms. Xiaomin Chen for initial technical support on this project. We also thank staffs from Chinese PLA 301 Hospital for providing cancer tissue samples, and all other members from Chen Lab for discussions and comments. Data analysis and computing resource was supported by Center for Big Data Research in Health (http://bigdata.ibp.ac.cn), Institute of Biophysics, Chinese Academy of Sciences. Author contributions Shuheng Wu: Conceptualization; Data curation; Formal analysis; Methodology; Validation; Visualization; Roles/Writing - original draft; Writing - review &editing. Erzhong Wu: Conceptualization; Data curation; Formal analysis; Methodology; Validation; Visualization; Roles/Writing - original draft; Writing - review &editing. Dongpeng Wang: Conceptualization; Data curation; Formal analysis; Methodology; Validation; Visualization; Roles/Writing - original draft; Writing - review &editing. Yiwei Niu: Conceptualization; Data curation; Formal analysis; Methodology; Validation; Software; Roles/Writing - original draft; Writing - review &editing. Haiyan Yue: Methodology; Resources. Dongdong Zhang: Methodology; Resources. Jianjun Luo: Conceptualization; Funding acquisition; Investigation; Project administration; Supervision; Roles/Writing - original draft; Writing - review &editing. Runsheng Chen: Conceptualization; Funding acquisition; Investigation; Project administration; Supervision.
Declaration of Interest Statement The authors declare that they have no competing interests.
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Figure captions
FIGURE 1 | Nuclear HRCEG decreased in gastric cancer. (A) HRCEG expression in GSE15459 and GSE79973 series from GEO database were normalized and compared between gastric cancer tissues and adjacent normal tissues. (B) qRT-PCR validation for gastric cancer tissues and adjacent normal tissues in 18 samples from external patient samples diagnosed with gastric cancer. (C) Schematic of the HRCEG gene locus and HRCEG RNA transcript obtained from our RACE results and annotation databases (NCBI RefSeq). (D) PCR amplification of HRCEG cDNA from BGC823 cells, indicating its transcript size. (E) The isolated nuclear and cytoplasmic total RNA was validated by qRT-PCR.
(F) 200 bp specific probe was used in FISH analysis to exhibit the nuclear-cytoplasmic location of HRCEG in BGC823 cells (HRCEG, red; nucleus, blue).
FIGURE 2 | Overexpression of HRCEG inhibited cell proliferation and promoted cell apoptosis. (A) pcDNA3.1- HRCEG and empty vector were transfected into MKN28 and BGC823 cells. 48 h later, cells were harvested and HRCEG expression was validated. (B)(C) MTS assays were performed in MKN28 cell (B) and BGC823 cell (C) with pcDNA3.1HRCEG and empty vector transfected to determine cell proliferation at 0 h, 24 h, 48 h, 72 h. (D)(E) At 48 h after transfection, MKN28 and BGC823 were stained and analyzed by flow cytometry. (E) Statistics of apoptotic cell were compared between pcDNA3.1- HRCEG and empty vector transfected cells. (F) Overexpression of HRCEG increased the expression level of Cleaved caspase3. *p<0.05; **p<0.01 ; ***p<0.001; ****p<0.001.
FIGURE 3 | Bioinformatics Analyses of HRCEG associated genes and expression levels in tumor samples. (A-F) Enrichment analysis results from gene set enrichment analysis (GSEA) of HRCEG correlated genes. (G) Expression levels of HRCEG in different types of gastric cancer, using dataset GSE15459. (H) Kaplan–Meier survival analysis of tumor samples grouped by median expression level of HRCEG, using TCGA-STAD dataset.
FIGURE 4 | Overexpression of HRCEG inhibited, whereas knockdown of HRCEG promoted cell invasion, migration and EMT of gastric cancer cells. (A) qRT-PCR analysis of HRCEG expression in BGC823 cells. (B)(C) Overexpression of HRCEG inhibited BGC823 cell invasion, and knockdown of HRCEG promoted BGC823 cell invasion. (D)(E) Overexpression of HRCEG inhibited BGC823 cell migration, and knockdown of HRCEG promoted BGC823 cell migration. (F) Scatter plot shows the expression of HRCEG and average expression of EMT-related genes, which were from MSigDB hallmark gene set "HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION". (G) Overexpression of HRCEG promoted the expression of E-cadherin, and inhibited the expression of Vimentin and Snail. (H) Knockdown of HRCEG inhibited the expression of E-cadherin and promoted the expression of Vimentin and Snail. *p<0.05; **p<0.01 ; ***p<0.001; ****p<0.001.
FIGURE 5 | HDAC1 associated to HRCEG gene region and regulated HRCEG RNA transcription in gastric cancer cells. (A) canSAR was used to associate HRCEG expression level with AJCC pathology in gastric cancer. (B) Binding enrichment analysis for HDAC1 at HRCEG loci in MKN28 and BGC823 cells. (C) siRNA-mediated loss-of-function of HDAC1 led to increased expression level of HRCEG in BGC823 cells. Overexpression of HDAC1 led to decreased expression level of HRCEG in BGC823 cells. *p<0.05; **p<0.01 ; ***p<0.001; ****p<0.001.
LncRNA HRCEG, Regulated by HDAC1, Inhibits Cells Proliferation and Epithelial-Mesenchymal-Transition in Gastric Cancer Shuheng Wu , Erzhong Wu , Dongpeng Wang , Yiwei Niu , Haiyan Yue , Dongdong Zhang , Jianjun Luo , Runsheng Chen PII: DOI: Reference:
S2210-7762(19)30558-7 https://doi.org/10.1016/j.cancergen.2019.12.007 CGEN 8588
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Cancer Genetics
Received date: Revised date: Accepted date:
13 October 2019 25 December 2019 25 December 2019
Please cite this article as: Shuheng Wu , Erzhong Wu , Dongpeng Wang , Yiwei Niu , Haiyan Yue , Dongdong Zhang , Jianjun Luo , Runsheng Chen , LncRNA HRCEG, Regulated by HDAC1, Inhibits Cells Proliferation and Epithelial-Mesenchymal-Transition in Gastric Cancer, Cancer Genetics (2020), doi: https://doi.org/10.1016/j.cancergen.2019.12.007
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Highlights
One LncRNA HRCEG inhibits cell proliferation and EMT process in gastric cancer.
HRCEG was significantly connected with gastric cancer related pathways and were also highly involved in the EMT processes.
The expression of HRCEG is regulated by HDAC1.
LncRNA HRCEG, Regulated by HDAC1, Inhibits Cells Proliferation and Epithelial-Mesenchymal-Transition in Gastric Cancer Shuheng Wu1,2+, Erzhong Wu1,2+, Dongpeng Wang1,2+, Yiwei Niu1,2, Haiyan Yue1, Dongdong Zhang1, Jianjun Luo1,2*, and Runsheng Chen1,2,3* 1 Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China 2 University of Chinese Academy of Sciences, Beijing, 100049, China 3 Guangdong Geneway Decoding Bio-Tech Co. Ltd, Foshan, 528316, China +
The authors wish it to be known that, in their opinion, the first three aothors should be regarded as Joint First Authors. The authors declare no competing financial interest. * Corresponding author at: Institute of Biophysics, Chinese Academy of Sciences, No. 15, Datun Road, Chaoyang District, Beijing 100101, China. Email addresses: [email protected] (J.Luo), [email protected] (R.Chen) Abstract Recently, a number of long noncoding RNAs (lncRNAs) have been reported to play significant roles in human tumorigenesis. However, only few gastric cancer related lncRNAs have been well characterized. Here, we identified one lncRNA HRCEG, whose expression was decreased in the gastric cancer tissues compared with adjacent normal tissues. Overexpression of HRCEG significantly promoted cell apoptosis and inhibited cell proliferation. Importantly, we demonstrated that HRCEG levels inversely correlated with EMT process and HRCEG was regulated by the histone deacetylase 1 (HDAC1) in gastric cancer. These findings suggest that HRCEG might be regulated by HDAC1 to inhibit gastric cancer progress and metastatic capability via EMT pathway. 1. Introduction Gastric cancer (cardia and noncardia gastric cancer combined) is an important cancer worldwide and is the fifth most frequently diagnosed cancer and the third leading cause of cancer death (Bray et al., 2018). Different from other gastrointestinal cancer, the incidence rate of gastric cancer is especially high in Eastern Asia (Rahman et al., 2014). Given the significant development of surgical technology and endoscopy, the 5-year mortality has been decreasing particularly for the early gastric cancer. However, for advanced gastric cancer, the survival rate is still less than 15% once metastasizes (Kang et al., 2014). In recent years, many molecular mechanisms of gastric cancer have been investigated, but the mechanism of gastric cancer progression such as proliferation, growth, migration, invasion and apoptosis is still limited and incomplete. Numerous studies have demonstrated that long non-coding RNAs may play important roles in gastric cancer progression (Song et al., 2017; Xu et al., 2017; Zong et al., 2019). Long non-coding RNAs (lncRNAs) are a class of ncRNAs longer than 200 nucleotides and
lack of protein-coding ability (Chen et al., 2014; Rinn and Chang, 2012). More recently, more than 172,216 lncRNA transcripts for human have been collected in our latest version of NONCODE database (Fang et al., 2018). LncRNAs act as activators, decoys, guides, or scaffolds for their interacting proteins, DNA and RNA (Yao et al., 2019). Increasing evidence has suggested that lncRNAs could play essential roles in almost all the biological processes, including stem cell maintenance (Wang et al., 2015; Wang et al., 2019), cell proliferation (Sun et al., 2017), cell apoptosis (Xu et al., 2015), migration (Hao et al., 2017), and epithelial-to-mesenchymal transition (EMT) in several cancers (Li et al., 2018). In the case of gastric cancer, however, there is still little knowledge about gastric cancer-specific lncRNAs, and the specific functional roles of these molecules are still unclear. Although several lncRNAs have been found to participate in gastric cancer to date, such as PVT1, MALAT1, DANCR, NEAT1, LINC00673 (Sawaki et al., 2018), we believe that there are still many unexplored differentially expressed lncRNAs in gastric cancer, especially their expression pattern and underlying regulation. In this study, we functionally characterized one lncRNA HRCEG (HDAC1 regulated RNA affecting Cell proliferation and EMT transition in Gastric cancer), a 3.8 kb antisense RNA of solute carrier family 25 member 25 (SLC25A25) gene. We found that expression of HRCEG was decreased in gastric cancer and overexpression of HRCEG inhibited gastric cancer cells proliferation and EMT process. The low expression of HRCEG in gastric cancer was regulated by HDAC1. These results suggest that further studies are needed to detail the functional mechanism of HRCEG in GC and HRCEG could be a new diagnostic biomarker and therapeutic target. 2. Materials and Methods 2.1. Tissue samples In this study, 18 pairs of gastric cancer samples were collected from Chinese PLA 301 Hospital. All samples contained pairs of gastric cancer tumor and adjacent normal tissue for qRT-PCR validation. This study was informed and obtained consent from each patient before their participation. All tissue samples were collected by surgical operation, fla sh freezing in liquid nitrogen and transferred to -80℃ freezer for storage. 2.2. Cell culture and Cell line transfection Human gastric cancer cell line, MKN28 and BGC823 were purchased from American Type Culture Collection (ATCC) and cultured with the suggestions in ATCC. Human gastric cancer cell line, MKN28 and BGC823 were cultured with DMEM medium(Life Technologies, 11995-065), supplemented with 10% fetal bovine serum(Life Technologies, 16000-044), 1% penicillin, and 1% streptomycin(Life Technologies, 10378-016 ) in a humidified 5% CO2 atmosphere at 37 ℃. For pcDNA3.1 and p-Enter plasmid transfection, cells were transfected with plasmids using Lipofectamine 2000 (Invitrogen, 11668-019) as suggested approaches. 2.3. Bioinformatics Analyses LncRNAs and mRNAs were identified from the gene expression data GSE15459 and GSE79973. Paired-t-test was used to obtain the differentially expressed genes using a Limma
package in R. The GseaPreRanked tool in GSEA v2.0 was used to perform GSEA of HRCEG. 2.4. qRT-PCR Total RNA was extracted by TRIzol reagent (Invitrogen, 15596-026) according to the manufacturer's instructions, and RNA was subjected to DNaseI (Invitrogen, AM2222) treatment. qRT-PCR was performed using the TransScript II Green One-Step qRT-PCR SuperMix kit (TransGen Biotech, AQ311-01) with 100 ng RNA as template in a 20 μl reaction volume on the Rotor-Gene® Q real-time cycler (Qiagen). The results were normalized with GAPDH, and the RNA relative expression was calculated by the ΔΔCt method. The primer sequences are listed in Supplemental Table 1. 2.5. shRNA-mediated interference and lentivirus packaging. shRNAs against HRCEG were designed using online software (https://rnaidesigner.thermofisher.com/rnaiexpress/design.do). Two shRNAs were designed, and a non-targeting, scrambled silencing RNA (scr) was used as a negative control. shRNAs targeting HRCEG and the scr hairpins were cloned into pSicoR-puro lentiviral vector. Virus packaging was carried out in 293T (ADCC) cell line after co-transfection of recombinant lentivirus expression plasmids for shHRCEG together with the packaging plasmids (PMD2G, psPAX2, Addgene) using Lipofectamine 2000 (Invitrogen, 11668-019). The virus was harvested at 48h after transfection, and infected BGC823 cell line for 24h. Then fresh medium with puromycin (2 μg/ml) was added to select for positive cells for additional 3 days incubation. The RNAi efficiency of HRCEG was checked by qRT-PCR analysis. 2.6. RNA interference siRNAs used to knock down HDAC1 mRNAs were designed online (https://rnaidesigner.thermofisher.com/rnaiexpress/design.do) and synthesized by the GenePharma company. A nonspecific siRNA used as the negative control (NC) was also purchased from the GenePharma Company. MKN28 and BGC823 cells were seeded in 6-well plates at a desired concentration for 12h and transfected with siRNA oligonucleotides with Lipofectamine 2000 according to the manufacturer's instructions. 2.7. Cell migration and invasion assay For knockdown and overexpression of HRCEG, BGC823 cells were infected with shHRCEG virus and transfected with pcDNA3.1-HRCEG plasmids, trypsinized and counted after 48 h. A total of 60,000 cells (for the migration assay) or 10,000 cells (for the invasion assay) in 100 μl serum-free medium were seeded in the upper chamber of Transwell plates (Corning, 3422) with (for invasion) or without (for migration) BD Matrigel matrix (BD, 356230) coating. 600 μl medium containing 20% FBS was added into the lower chambers as the chemotactic factor. After 10h cell culture at 37℃ and 5% CO2, the non-migrating or non-invading cells in upper chamber were removed, and the migrating or invading cells in lower chamber were stained with 0.1% crystal violet for 20min, then washed with water, air dried and photographed using Nikon microscope imaging systems. The migrating and invading cells were counted and averaged from images of six random fields (magnification was 20× or 40×) for differential analysis.
2.8. Cell proliferation and apoptosis Cell proliferation ability was examined using a Cell Proliferation Reagent Kit I (MTS; Promega, G3580). MKN28 and BGC823 cells were harvested and resuspended in PBS. Then the cells were stained with Dead Cell Apoptosis Kit with Annexin V Alexa Fluor™ 488 & Propidium Iodide (PI) (Invitrogen, V13245) as instructions and analyzed by Flow Cytometry (BD FACS Calibur, USA). 2.9. Subcellular fractionation The nuclear and cytoplasmic fractions of MKN28 and BGC823 cells were isolated with the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo, 78833). The nuclear and cytoplasmic location of HRCEG was assessed by qRT-PCR on RNA isolated from the subcellular fractions. GAPDH and ACTB were served as the cytosolic controls while U1 and MALAT1 was used as the nuclear control. 2.10. Western Blot Western blotting was performed as standard protocols. And the primary antibodies used in this study were obtained as follows: ACTB (Proteintech, 66009-1-Ig ), Snail (CST,C1503), Vimentin (Invitrogen, MA1-10459), E-cadherin (CST, 144725), Cleaved CASP3 (ABGENT, Asp175). 2.11. RNA fluorescence in situ hybridization In vitro transcription of DIG-labeled 200bp antisense HRCEG RNA probe was conducted using T7 RNA polymerases (Roche) and DIG RNA Labeling Mix (Roche). Probe template was amplified with ExPfu PCR StarMix (Genstar, A063-01). Synthesized probes were purified using RNeasy® mini kit (Qiagen). RNA FISH was performed as described in (http://wmc.rodentia.com/docs/Big_In_Situ.html) with minor modifications. Briefly, after fixed with 4% formaldehyde for 10min, cells cultured on coverslips were permeabilized with 1x PBS/0.5% Triton X-100 followed by acetylation and pre-hybridization. Hybridization was performed with probes at a concentration of 1µg/ml at 65°C overnight. For detection of dig labelled probes, the coverslips were incubated with mouse monoclonal Anti-Digoxigenin antibody (Abcam) (1:200) for 1h at room temperature, and signals were visualized by TSA Fluorescein System (PerkinElmer, 744001). The coverslips were then washed, mounted with SlowFade Gold Antifade Reagent (Invitrogen, p36930) and observed with an Olympus FV1200 confocal microscope. Primer sequences were shown in Supplemental Table 1. 2.12. 5′ and 3′ RACE. 5′ and 3′ RACE of HRCEG were performed using the SMARTer® RACE 5’/3’ Kit (Takara, 634858) as recommended by the manufacturer. RACE PCR products were obtained using ExPfu PCR StarMix (Genstar, A063-01), the appropriate gene-specific primers are summarized in Supplemental Table 1. RACE PCR products were separated on a 1% agarose gel. Gel products were extracted with the Gel and PCR Clean-Up System (Promega, A9282), cloned into the pGEM-T Vector Systems I (Promega, A3600), and sequenced bi-directionally using the M13 forward and reverse primers by Sanger sequencing at Sangon Biotech. At least five colonies were
sequenced for every RACE PCR product that was gel purified. 2.13. Chromatin immunoprecipitation (ChIP) and quantitative analysis HDAC1 antibody (abcam, ab7028) was used to perform the ChIP assay in MKN28 and BGC823 cells. ChIP assays were carried out as described previously (Nelson et al., 2006). Briefly, MKN28 and BGC823 cells were cross-linked by 1% formaldehyde for 10min and the reaction was quenched with 250 mM glycine. After washing with cold DEPC-PBS, fixed MKN28 and BGC823 cells were pelleted and resuspended in 1ml sonication buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA). Cell suspension was sonicated in ice bath to generate chromatin fragments of ~500bp in length. Chromatin lysate was cleared by centrifugation and diluted to 10 folds with dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, pH8.0). 1% volume of the sample was taken as input DNA and 4 µg antibodies against HDAC1 were added to the samples and rotated at 4 °C overnight. After hybridization, 50 µl of Protein A/G beads (Thermo Fisher, 88802) was added to each hybridization reaction. Beads were captured by DynaMag-15 magnetic strip and washed with 1 ml low salt buffer (0.1% SDS, 20 mM Tris-HCl, pH 8.0, 1% Triton X-100, 150 mM NaCl) once, high salt buffer (0.1% SDS, 20 mM Tris-HCl, pH 8.0, 1% Triton X-100, 500mM NaCl) once, LiCl buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.25 mM LiCl, 0.1% NP-40, 1% deoxycholate sodium) once and TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) twice. 100 µl Elution buffer (0.1 M NaHCO3, 1% SDS and proteinase K) and 8 µl 5M NaCl was used to reverse cross-linking of DNA–protein complexes at 65 °C for 4–5h. DNA was purified by phenol-chloroform extraction and subjected to qPCR. All primers used in this study are listed in Supplemental Table 1. 2.14. Statistical Analysis All statistical analyses were performed using Graphpad Prism 7.0 statistical software (California, US). Experimental data are shown as the means ± SD, all experiments were conducted at least three times. Significance was determined using the Student's t-test or two-way ANOVA: *, p<0.05; **, p<0.01 and ***, p<0.001. 2.15. Data Availability All data generated during this study are available from the corresponding author on reasonable request.
3. Results 3.1. HRCEG significantly under-expressed in gastric cancer Several lncRNAs are found performing very important functions in alimentary canal cancers (Chen et al., 2018; Guo et al., 2018; Sun et al., 2017; Yan et al., 2017), especially in colorectal cancer and gastric cancer. In a course of studying the transcriptional profiling analysis and functional prediction of long noncoding RNAs in cancer (Yuan et al., 2016) as well as transcriptional signature analyses for gastric cancer (data not shown), we also noticed one gastric cancer specific differentially expressed unknown lncRNA, herein termed as HRCEG. To further investigate the roles of HRCEG in gastric cancer, we first analyzed two additional microarray data from NCBI GEO database (i.e. GSE15459 and GSE79973). We found that HRCEG significantly decrease in gastric cancer compared with paired-adjacent non-tumor (Figure 1A). Furthermore, we evaluated expression pattern of HRCEG using 18 pairs of gastric cancer tissues and adjacent normal tissues from patient samples (Figure 1B). To characterize HRCEG in detail, we used RACE assay to identify the full sequence of HRCEG in MKN28 cell line based on the sequence archived in RefSeq database of NCBI. Sequence with 3.8kb in length was successfully amplified and aligned (Figure 1C), which is a little longer than archived in the RefSeq database. Full length PCR amplification with primers designed according to RACE produced sequence also yielded a product with size similar to the RACE result of HRCEG (Figure 1D). Furthermore, as localization of lncRNA reflects typical function categories of RNA, next, we examined the subcellular localization of HRCEG in MKN28 cell line by nuclear-cytoplasmic fractionation (Figure 1E) and RNA fluorescence in situ hybridization (FISH) (Figure 1F), and data clearly indicated that the majority of HRCEG transcript resides in nucleus. These results indicated that we identified one nuclear localized lncRNA HRCEG, whose expression was potentially repressed in the gastric cancer tissues compared to nearby normal tissues. 3.2. HRCEG regulate gastric cancer cell proliferation and apoptosis To further investigate the physiological roles of HRCEG in gastric cancer, we performed gain-of function studies in gastric cancer cell lines MKN28 and BGC823. As shown in Figure 2A, RT-qPCR show that the pCDNA3.1-HRCEG vector could significantly increase the expression of HRCEG. Then, we measured cell viability in both HRCEG high expressed BGC-823 and MKN28 cell lines by MTS assay. Compared with control cells transfected with empty vector, overexpression of HRCEG inhibited cell proliferation (Figures 2B, C). Additionally, we also examined the effect of HRCEG on apoptosis by Annexin-V and FACS analysis, and found that cell apoptosis significantly increased upon the up-regulation of HRCEG (Figures 2D, E). Consistently, we also observed that overexpression of HRCEG resulted in more Caspase3 cleavage compared to control cells (Figure 2F). These results indicated that HRCEG might inhibit gastric cancer progress by regulating cell proliferation and apoptosis. To assess the molecular pathways that HRCEG may be involved in, we performed Gene Set Enrichment Analysis (GSEA) of HRCEG associated gene sets by Pearson correlation coefficients between expression profiles of mRNA and HRCEG (GEO15459). The results revealed that the
gene sets enriched in gastric cancer progress, EMT, cell apoptosis, cell cycle and cancer related pathways (Figures 3A-F). The correlation between HRCEG and the clinical features of cancer patients also showed that HRCEG expression levels was lower in invasive subtype (P = 0.001) (Figure 3G). Kaplan-Meier survival analysis supported that lower HRCEG expression correlated with worse overall survival, indicating that decreased expression of HRCEG is correlated with gastric cancer progression and poor prognosis (Figure 3H). 3.3. HRCEG suppresses EMT process in gastric cancer cells As our results showed that HRCEG may function as a tumor suppressor in gastric cancer, we next investigated whether HRCEG was involved in the EMT process. The GSEA of the dataset GSE15459 indicated a high correlation between HRCEG and EMT process (Figure 3B). To study this further, we performed the transwell assays to measure cell metastatic capability. As shown in Figures 4A, RT-qPCR show that designed shRNAs could significantly decreased the expression of HRCEG. HRCEG knock-down by specific shRNA treatment increased the invasive capacity of BGC823 cells, whereas, overexpression of HRCEG inhibited the invasion of BGC823 cells (Figures 4B, C). In the migration assay, we also found that down-regulation of HRCEG significantly reduced the migration ability of BGC823 cells and up-regulation of HRCEG promoted BGC823 cell migration. Bioinformatics analysis showed that HRCEG had a significantly negative correlation with EMT related genes in GSE15459 dataset (Figure 4F). Then, we detected the expression level changes of the EMT markers following HRCEG knockdown and overexpression by Western blotting. As shown in Figure 4G, HRCEG overexpression remarkably increased the expression of E-cadherin but decreased expressions of mesenchymal-related genes, including Snail and Vimentin. Consistently, we also detected that lower expression of E-cadherin and higher expression of Snail and Vimentin upon down-regulation of HRCEG (Figure 4H). Taken together, these data show that HRCEG influence gastric cancer progression by affecting the EMT processes. 3.4. HDAC1 suppressed HRCEG transcription in gastric cancer cells Previous reports demonstrated that epigenetic modification could regulate lncRNAs transcription in cancer cells such as methylation and acetylation (Dong et al., 2018; Rokavec et al., 2017; Zhang et al., 2017). It is known that histone deacetylase 1 (HDAC1) could erase the acetylation status of core histone, regulate genes transcription and play important regulatory roles in cancer cells (Weichert et al., 2008). HDAC1 expression has been reported to be up-regulated in gastric cancer (Mutze et al., 2010), and we found HRCEG expression level was associated with AJCC pathology in gastric cancer using the canSAR 1 (Figure 5A). Genes correlated with HRCEG shown in GSEA analysis were also enriched for HDAC1 targets, which suggests the possibility that HRCEG is regulated by HDAC1 in gastric cancer cells. Using ChIP-qPCR assays, we found that HDAC1 could enrich to the HRCEG gene region (Figure 5B). To investigate the relationship between HRCEG and HDAC1, we knockdown and up-regulate HDAC1 in gastric cancer cells (Figures 5D, E). The expression of HRCEG increased upon knockdown of HDAC1 and decreased upon overexpression of HDAC1 (Figures 5D, E). In conclusion, these data confirmed that HRCEG is regulated by HDAC1 and may explain the reason of low expression of HRCEG in gastric cancer.
4. Discussion LncRNAs are emerging components that are recognized to play critical roles in cancer development and progression. Recently, some lncRNAs have been found to participate in gastric cancer tumorigenesis. LncRNAs function by impacting proliferation, metastasis, migration, and epithelial-to-mesenchymal transition (EMT). Here, we demonstrated that lncRNA HRCEG, which is mainly localized in the nucleus and expression level significantly decreased in gastric cancer tissues compared with adjacent normal tissues and cell lines, including BGC823 and MKN28. Thus, expression of HRCEG may be required to repress gastric cancer tumorigenesis and progression. It has been shown that lncRNAs play key roles in regulation of the malignant phenotypes of cancer cells. In this study, we found that overexpression of HRCEG inhibited gastric cancer cell proliferation, and induced cell apoptosis. Our findings suggest that HRCEG may function as a tumor suppressor lncRNA in gastric cancer and potentially be considered as a novel prognostic indicator for gastric cancer. We further determined the expression levels of apoptosis-related proteins. Results show that the inhibition on HRCEG promoted the accumulation of apoptotic protein caspase-3. Caspase-3 is a frequently activated death protease, catalyzing the specific cleavage of many key cellular proteins (Porter and Janicke, 1999). Activation of pro-caspase-3 is a central event in the execution phase of apoptosis and appears to serve as the convergence point of different apoptotic signaling pathways. Increasing evidence has demonstrated a potential role of lncRNAs in gastric cancer metastasis by influence the EMT process. Our GSEA data illustrated that HRCEG was significantly connected with gastric cancer related pathways and were also highly involved in the EMT processes. Furthermore, our cell invasion and migration assays revealed that HRCEG indeed play regulatory roles on gastric cancer metastasis. The feature of EMT occurrence is that the epithelial marker E-cadherin is down-regulated and mesenchymal marker, like Snail and Vimentin, is up-regulated. To demonstrate whether HRCEG affected EMT markers, we detected the molecular marker levels of EMT and showed the inhibition of HRCEG on EMT. Histone Deacetylase 1 (HDAC1) is tightly correlated with gastric cancer, and its high expression has been shown to correlate with advanced stage, uncontrolled tumor cell proliferation and poor prognosis in gastric cancer, which implies that HDAC1 has a critical role in carcinogenesis. HDAC1 was recently identified as an epigenetic regulator through modulation of FENDRR transcriptional activation, and HDAC1 contributed to the decreased expression of FENDRR in gastric cancer cells. Besides, FENDRR suppresses gastric cancer cell metastasis in vitro and in vivo, and a negative correlation between fibronectin (FN)1 and FENDRR expression was reported. Thus, we further determined whether HDAC1 is a regulator of HRCEG disorder expression in gastric cancer cells. Interestingly, our results confirmed that HDAC1 down-regulates HRCEG expression in gastric cancer, and decreased HRCEG expression induces Snail and Vimentin expression. Our findings revealed that HRCEG inhibits cell proliferation and EMT process in gastric
cancer. The expression of HRCEG is regulated by HDAC1. More works are needed to characterize the underlying mechanism how HRCEG serves as a regulatory factor in the gastric cancer. Funding This work was supported by grants from the National Key Research and Development Program of the Ministry of Science and Technology of China (2017YFC0907503 and 2018YFA0106901), as well as grant from the National Natural Science Foundation of China (31520103905). Conflict of interest The authors declare that they have no competing interests. Acknowledgements We are very grateful to Dr. Hangcheng Huang and Ms. Xiaomin Chen for initial technical support on this project. We also thank staffs from Chinese PLA 301 Hospital for providing cancer tissue samples, and all other members from Chen Lab for discussions and comments. Data analysis and computing resource was supported by Center for Big Data Research in Health (http://bigdata.ibp.ac.cn), Institute of Biophysics, Chinese Academy of Sciences. Author contributions Shuheng Wu: Conceptualization; Data curation; Formal analysis; Methodology; Validation; Visualization; Roles/Writing - original draft; Writing - review &editing. Erzhong Wu: Conceptualization; Data curation; Formal analysis; Methodology; Validation; Visualization; Roles/Writing - original draft; Writing - review &editing. Dongpeng Wang: Conceptualization; Data curation; Formal analysis; Methodology; Validation; Visualization; Roles/Writing - original draft; Writing - review &editing. Yiwei Niu: Conceptualization; Data curation; Formal analysis; Methodology; Validation; Software; Roles/Writing - original draft; Writing - review &editing. Haiyan Yue: Methodology; Resources. Dongdong Zhang: Methodology; Resources. Jianjun Luo: Conceptualization; Funding acquisition; Investigation; Project administration; Supervision; Roles/Writing - original draft; Writing - review &editing. Runsheng Chen: Conceptualization; Funding acquisition; Investigation; Project administration; Supervision.
Declaration of Interest Statement The authors declare that they have no competing interests.
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Figure captions
FIGURE 1 | Nuclear HRCEG decreased in gastric cancer. (A) HRCEG expression in GSE15459 and GSE79973 series from GEO database were normalized and compared between gastric cancer tissues and adjacent normal tissues. (B) qRT-PCR validation for gastric cancer tissues and adjacent normal tissues in 18 samples from external patient samples diagnosed with gastric cancer. (C) Schematic of the HRCEG gene locus and HRCEG RNA transcript obtained from our RACE results and annotation databases (NCBI RefSeq). (D) PCR amplification of HRCEG cDNA from BGC823 cells, indicating its transcript size. (E) The isolated nuclear and cytoplasmic total RNA was validated by qRT-PCR.
(F) 200 bp specific probe was used in FISH analysis to exhibit the nuclear-cytoplasmic location of HRCEG in BGC823 cells (HRCEG, red; nucleus, blue).
FIGURE 2 | Overexpression of HRCEG inhibited cell proliferation and promoted cell apoptosis. (A) pcDNA3.1- HRCEG and empty vector were transfected into MKN28 and BGC823 cells. 48 h later, cells were harvested and HRCEG expression was validated. (B)(C) MTS assays were performed in MKN28 cell (B) and BGC823 cell (C) with pcDNA3.1HRCEG and empty vector transfected to determine cell proliferation at 0 h, 24 h, 48 h, 72 h. (D)(E) At 48 h after transfection, MKN28 and BGC823 were stained and analyzed by flow cytometry. (E) Statistics of apoptotic cell were compared between pcDNA3.1- HRCEG and empty vector transfected cells. (F) Overexpression of HRCEG increased the expression level of Cleaved caspase3. *p<0.05; **p<0.01 ; ***p<0.001; ****p<0.001.
FIGURE 3 | Bioinformatics Analyses of HRCEG associated genes and expression levels in tumor samples. (A-F) Enrichment analysis results from gene set enrichment analysis (GSEA) of HRCEG correlated genes. (G) Expression levels of HRCEG in different types of gastric cancer, using dataset GSE15459. (H) Kaplan–Meier survival analysis of tumor samples grouped by median expression level of HRCEG, using TCGA-STAD dataset.
FIGURE 4 | Overexpression of HRCEG inhibited, whereas knockdown of HRCEG promoted cell invasion, migration and EMT of gastric cancer cells. (A) qRT-PCR analysis of HRCEG expression in BGC823 cells. (B)(C) Overexpression of HRCEG inhibited BGC823 cell invasion, and knockdown of HRCEG promoted BGC823 cell invasion. (D)(E) Overexpression of HRCEG inhibited BGC823 cell migration, and knockdown of HRCEG promoted BGC823 cell migration. (F) Scatter plot shows the expression of HRCEG and average expression of EMT-related genes, which were from MSigDB hallmark gene set "HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION". (G) Overexpression of HRCEG promoted the expression of E-cadherin, and inhibited the expression of Vimentin and Snail. (H) Knockdown of HRCEG inhibited the expression of E-cadherin and promoted the expression of Vimentin and Snail. *p<0.05; **p<0.01 ; ***p<0.001; ****p<0.001.
FIGURE 5 | HDAC1 associated to HRCEG gene region and regulated HRCEG RNA transcription in gastric cancer cells. (A) canSAR was used to associate HRCEG expression level with AJCC pathology in gastric cancer. (B) Binding enrichment analysis for HDAC1 at HRCEG loci in MKN28 and BGC823 cells. (C) siRNA-mediated loss-of-function of HDAC1 led to increased expression level of HRCEG in BGC823 cells. Overexpression of HDAC1 led to decreased expression level of HRCEG in BGC823 cells. *p<0.05; **p<0.01 ; ***p<0.001; ****p<0.001.