A novel long noncoding RNA HOXC-AS3 mediates tumorigenesis of gastric cancer by binding to YBX1
Recently, increasing evidence shows that long noncoding RNAs (lncRNAs) play a significant role in human tumorigenesis. However, the function of lncRNAs in human gastric cancer remains largely unknown.
By using publicly available expression profiling data from gastric cancer and integrating bioinformatics analyses, we screen and identify a novel lncRNA, HOXC-AS3. HOXC-AS3 is significantly increased in gastric cancer tissues and is correlated with clinical outcomes of gastric cancer. In addition, HOXC-AS3 regulates cell proliferation and migration both in vitro and in vivo. RNA-seq analysis reveals that HOXC-AS3 knockdown preferentially affects genes that are linked to proliferation and migration. Mechanistically, we find that HOXC-AS3 is obviously activated by gain of H3K4me3 and H3K27ac, both in cells and in tissues. RNA pull-down mass spectrometry analysis identifies that YBX1 interacts with HOXC-AS3, and RNA-seq analysis finds a marked overlap in genes differentially expressed after YBX1 knockdown and those transcriptionally regulated by HOXC-AS3, suggesting that YBX1 participates in HOXC-AS3-mediated gene transcriptional regulation in the tumorigenesis of gastric cancer.
Together, our data demonstrate that abnormal histone modification-activated HOXC-AS3 may play important roles in gastric cancer oncogenesis and may serve as a target for gastric cancer diagnosis and therapy.
KeywordsHistone modification HOXC-AS3 YBX1 GC
Gastric cancer is one of the leading causes of cancer-related deaths worldwide and the most common gastrointestinal malignancy in East Asia [1, 2]. Gastric cancer is diagnosed at an advanced stage accompanied by malignant proliferation in most patients, and the prognosis for advanced stage patients remains very poor . Therefore, additional research is needed to discover and develop effective biomarkers and targets for gastric cancer diagnosis and treatment.
To date, gastric cancer research has mainly focused on the deregulation of protein-coding genes to identify oncogenes and tumor suppressors that could serve as diagnostic and therapeutic targets. However, protein-coding sequences occupy less than 2% of the human genome [4, 5]. LncRNAs are operationally defined as RNA transcripts that are > 200 nt with limited protein coding potential , which have been shown to play a key role in tumorigenesis, including GC [7, 8, 9]. Many studies found that lncRNAs could play an important role in regulating gene expression by different mechanisms, including chromatin modification, and transcriptional and posttranscriptional processing [10, 11, 12]. For example, HOTAIR is involved in the transcriptional repression of HOX loci and promotes breast metastasis by binding to PRC2 (Polycomb Repressive Complex) . However, the biological functions of lncRNAs in the control of GC tumorigenesis are not well characterized. Therefore, a better understanding of the role of lncRNAs underlying GC progression will enrich the understanding of the molecular mechanisms of GC carcinogenesis and provide information for improving the diagnosis and treatment of GC.
In our present study, we identified the full sequence of HOXC-AS3 and found that gain of H3K4me3 and H3K27acetylation could activate the expression of HOXC-AS3, both in cells and in tissues. HOXC-AS3 was also significantly upregulated in GC tissues compared with the corresponding nontumor tissues and may serve as an independent predictor for the overall survival in GC. In addition, HOXC-AS3 regulated cell proliferation and migration both in vitro and in vivo. RNA-seq analysis for whole transcriptome studies indicates an important role for HOXC-AS3 in the tumorigenesis of GC, and the activated function of HOXC-AS3 was mediated, in part, by interaction with YBX1. These results suggest that further studies to identify nonprotein-coding genes that contribute to oncogenesis are necessary for elucidating the complex genetic rewiring that is driven by HOXC-AS3 in GC.
Identification of HOXC-AS3 by analyzing gastric cancer RNA-expression profiling data
Gain of H3K4me3 and H3K27 acetylation-activated HOXC-AS3 is upregulated in human GC tissues and correlates with poor prognosis
For further study, we first performed rapid amplification of cDNA ends (RACE) to identify the full sequence of HOXC-AS3 in BGC-823 cells according the sequence archived in the RefSeq database of NCBI (440 bp, with poly (A) tail, Fig. 2b). To validate the expression results from high-throughput data, as shown in Fig. 2c, the HOXC-AS3 expression level in tumor tissues was significantly higher in 112 pairs of GC tumor tissues compared with adjacent normal tissues (P < 0.001). One tumor tissue showed an upregulation of HOXC-AS3 greater than 521-fold relative to normal tissue. Next, we explored the correlation between HOXC-AS3 expression and the clinicopathological factors of patients with GC. The result showed that the HOXC-AS3 level was associated with TNM stage. Patients with advanced TNM stage (III/IV) were associated with higher HOXC-AS3 expression, whereas patients with local TNM stage (I/II) were associated with a lower HOXC-AS3 level (27.1317 ± 80.26254 vs 4.7483 ± 3.02402, P = 0.046) (Fig. 2d). Furthermore, we divided the samples into relatively high (above the mean, n = 56) and relatively low (below the mean, n = 56) HOXC-AS3 expression groups according to the median value of HOXC-AS3 levels. A chi-square test was then performed to evaluate clinicopathological factors between the two groups. As shown in Additional file 1: Table S1, the relative HOXC-AS3 level was also correlated with histological grade (P = 0.002), tumor invasion depth (P = 0.008), lymph node metastasis (P = 0.035), and TNM stage (P = 0.002). No relationship between HOXC-AS3 expression and other clinical factors, such as sex (male, female) and patient age (≤ 60, > 60), was found in our study.
To determine the relationship between HOXC-AS3 expression and GC patient prognosis, we evaluated the correlation between HOXC-AS3 expression and clinical outcomes. Kaplan–Meier analysis and log-rank test were used to evaluate the effects of HOXC-AS3 expression and the clinicopathological characteristics on overall survival (OS). The median survival time for low HOXC-AS3 expression groups was 34 months, whereas for high HOXC-AS3 expression groups, it was only 20 ± 1.357 months. As shown in Fig. 2e, overexpression of HOXC-AS3 predicted a poor prognosis in patients with GC (P = 0.004). Similarly, the correlation between HOXC-AS3 expression levels and the survival of GC patients was also supported by Kaplan–Meier Plotter analysis (http://kmplot.com/analysis/, detailed steps were described in Additional file 8: Supplementary Methods), which indicated that higher HOXC-AS3 expression correlated with worse OS, using publicly available chip data from 631 GC patients (Fig. 2f).
Then, univariate and multivariate survival analyses (Cox proportional hazards regression model) were performed. Univariate analysis identified two prognostic factors: TNM stage and HOXC-AS3 expression. Multivariate analysis further revealed that HOXC-AS3 expression was an independent predictor for overall survival in patients with GC (P < 0.001), as well as TNM stage (P = 0.023) (Additional file 2: Table S2).
To explore the mechanism of high expression of HOXC-AS3 in GC, firstly, by using the UCSC Genome Bioinformatics Site (http://genome.ucsc.edu/), we found high enrichment and overlaps of H3K4me3 and H3K27Ac peaks at the promoter region of HOXC-AS3 (Fig. 2g, H3K4me3 and H3K27Ac, two markers of active promoters). Using ChIP assays, we found gain of H3K4me3 and H3K27Ac in cancer tissues compared with normal tissues (n = 4) at the promoter of HOXC-AS3. We also observed the gain of H3K4me3 and H3K27Ac in GC cells (BGC-823) compared with normal human esophageal epithelial cells (GES-1) at the promoter of HOXC-AS3 (Fig. 2g). Taken together, these data confirm that HOXC-AS3 is frequently increased in GC. Abnormal histone modification, gain of histone sites H3K4me3 and H3K27Ac of the promoter may partially account for the significant activation of HOXC-AS3.
HOXC-AS3 regulates GC cell proliferation and migration in vitro
HOXC-AS3 regulates GC cell proliferation and migration in vivo
To validate the effects of HOXC-AS3 on cell metastasis in vivo, BGC-823 cells stably transfected with sh-HOXC-AS3 or control vector were injected into the tail veins of nine mice. Metastatic nodules on the surface of the lungs were counted after 7 weeks. Ectopic knockdown of HOXC-AS3 reduced the number of metastatic nodules compared with the control group (Fig. 4b). This difference was further confirmed following examination of the entire lungs and by hematoxylin and eosin (HE) staining of lung sections (Fig. 4b). Our in vivo data, therefore, complemented the results of the functional in vitro studies involving HOXC-AS3.
HOXC-AS3 interacts with YBX1
A large set of genes that are linked to cell proliferation and cell migration were coregulated by interaction of HOXC-AS3 and YBX1
HOXC-AS3 regulates the expression of HDAC5
Before the discovery of noncoding RNAs, explorations for cancer drivers focused on protein-coding genes that resided in recurrent alterations in cancer genomes. However, the newly discovered lncRNAs have emerged as important players in cellular development and human diseases, especially in cancer. In the present study, utilizing publicly available lncRNA expression profiling data of gastric cancer and integrating analyses of TCGA data, we screened and identified a novel lncRNAs HOXC-AS3. The high expression of HOXC-AS3 in GC patients was positively correlated with advanced TNM stage. Moreover, high HOXC-AS3 expression in GC tissues was associated with a poor prognosis and could be an independent prognostic indicator. In addition, GTEx data (https://www.gtexportal.org/) showed that HOXC-AS3 had lower basal expression in normal GC tissues (Additional file 3: Figure S1B). This further demonstrates an important role of HOXC-AS3 in the carcinogenesis of GC. These results and our functional evidence for HOXC-AS3 suggested that HOXC-AS3 might exhibit an important role in GC progression.
HOXC-AS3, which is located at chromosome 12q13.13, was an antisense transcript of HOXC10. HOX genes are essential for morphogenesis and development [31, 32]. The dysregulation of HOX gene expression has been shown in many diverse cancers [33, 34], and lncRNA generation in HOX genes may play important roles in tumorigenesis. For example, as a well-known lncRNA, HOTAIR, also located at chromosome 12q13.13, is an antisense transcript of HOXC11 and was an oncogenic lncRNA in many different types of cancer . Our previous study also found that a lncRNA of the HOX gene family, HOXA11-AS, could play an important role in GC tumorigenesis . In our present study, we found a novel lncRNA in the HOX genes family. Our results revealed that gain of H3K4me3 and H3K27Ac activation of the promoter also partly contributed to activation of HOXC-AS3 in GC, both in cells and tissues. Similar to protein coding transcripts, the transcription of lncRNAs is subject to typical epigenetics-mediated and transcription factor-mediated regulation. For example, the lncRNA MEG3 was lost in tumors due to an increase in CpG methylation within the promoter . Histone deacetylase3-suppressed the lncRNA LET in hepatocellular carcinoma by reducing the histone acetylation-mediated modulation of the promoter region .
In our study, we found that inhibition of HOXC-AS3 repressed GC proliferation and migration both in vitro and in vivo. RNA-seq found that knockdown of HOXC-AS3 affected key cancer-related genes, such as p21, FAS, and CCND1. Mechanistic investigations found that HOXC-AS3 could bind to YBX1, but not affect YBX1 expression. These results indicated that HOXC-AS3 may participate in the tumorigenesis of GC through the transcriptional regulation of other genes via binding to YBX1 in trans. To probe the HOXC-AS3-associated pathway on an unbiased basis in the tumorigenesis of GC, RNA-Seq assays were used after simultaneous knockdown HOXC-AS3 and YBX1. Notably, loss of HOXC-AS3 in GC cells recapitulated the phenotype observed after YBX1 knockdown. In addition, a significant fraction of the genes regulated by HOXC-AS3 loss were similarly regulated by loss of YBX1. Thus, HOXC-AS3 may act, in part, by regulating the interaction between YBX1 and the promoter of target genes, although HOXC-AS3 likely interacts with other RNA-binding proteins that will need to be identified to fully understand its molecular function. YBX1 was reported to play a role in regulating cell signaling, transcription, and tumorigenesis . Furthermore, YBX1 serves as a transcriptional activator and regulates much gene transcription . YBX1 was a protein with a nucleic acid-binding common domain in the gene promoter, CCAAT-box, which is a high consensus sequence in eukaryotes. We found that YBX1 is overactive in GC and knockdown of YBX1 inhibits the proliferation of GC cells. Our results showed that HOXC-AS3 could bind to YBX1, thus transcriptionally regulating a large set of genes that are linked to cell proliferation and cell migration in gastric cancer cells, such as MMP7, WNT10B, and HDAC5, thus promoting GC cell proliferation and migration.
The HDAC5 gene is a member of the histone deacetylase (HDAC) from a family of enzymes. Histone acetylation and deacetylation play important roles in chromatin remodeling and gene expression. An imbalance of these reactions leads to the growth, migration, and apoptosis of cancer cells. Histone deacetylase (HDAC) inhibitors were shown to have antitumor effects in clinical trials [39, 40]. Over-activation of HDAC5 was found in many different types of cancer [41, 42, 43]. A previous study showed that HDAC5 was induced in gastric cancer cells . Here, we also provide evidence for high expression of HDAC5 in gastric cancer, and knockdown of HDAC5 inhibits the proliferation of GC cells. We also found that the transcriptional activation of HDAC5 is partly mediated by HOXC-AS3 in the tumor progression of GC through binding to YBX1, thus facilitating GC cell proliferation and migration. In addition to HDAC5, there are many other important genes related to tumorigenesis, and they are also regulated by HOXC-AS3 in a similar manner.
In summary, abnormal histone modification-mediated activation of a novel lncRNA HOXC-AS3 promotes GC cell proliferation and migration through transcriptional activation of a large set of genes through an interaction with YBX1. Our data reveal a role for HOXC-AS3 in GC tumorigenesis and may provide a strategy for using HOXC-AS3 as a potential biomarker and a therapeutic target for patients with GC (Fig. 8e).
Tissue collection and ethics statement
A total of 112 patients in this study underwent resection of the GC at The Affiliated Jiangyin Hospital of Southeast University Medical College, affiliated Xuzhou Central Hospital of Southeast University Medical College. The study was approved by the Medical Ethical Committee of Southeast University Medical College (Nanjing, Jiangsu, PR China), and it was performed in compliance with the Helsinki Declaration. All patients have given written informed consent for publication. The clinicopathological characteristics of the GC patients are summarized in Additional file 1: Table S1.
Gastric cancer RNA-expression data retrieval and analysis
Microarray data analysis
Microarray datasets from the GEO database were used to test HOXC-AS3 differential expression. Raw microarray data was downloaded from GEO including GSE50710 and GSE58828. Then, the raw microarray data were normalized and z-score-transformed using RMAExpress (http://www.rmaexpress.bmbolstad.com/). RNA-Seq data (from TCGA) of lncRNAs of gastric cancer were from TANRIC (http://ibl.mdanderson.org/tanric/_design/basic/index.html) .
RACE (rapid amplification of cDNA ends)
5′-RACE, 3′-RACE, and full-length amplification of HOXC-AS3 were performed using the SMART RACE cDNA Amplification Kit (Cat. 634858, Clontech, Palo Alto, CA, USA) according to the manufacturer’s instructions. The gene-specific primers used for RACE analysis were presented in Additional file 7: Table S6.
Transfection of cell lines
LNA-ASO (Locked Nucleic Acid, antisense oligonucleotide) targeting HOXC-AS3 and negative control LNA-ASO were designed and synthesized by Exiqon (Exiqon, Vedbaek, Denmark). GC cells were transfected with the LNA-ASOs using Oligofectamine transfection reagent (RNAi MAX, Invitrogen) according to the manufacturer’s instructions. Cells were harvested for analyses 48 h after transfections. The sequences of ASO were listed in Additional file 7: Table S6. The sequences for siRNAs were listed in Additional file 7: Table S6. Scrambled negative control siRNA was purchased from Invitrogen (Invitrogen, CA, USA). The interference target sequence of YBX1 was acquired according to a previous study . The HDAC5 siRNA was from Santa Cruz (sc-35542). The plasmid was transfected into GC cells using the X-tremeGENE™ HP DNA Transfection Reagent (Roche) according to the manufacturer’s instructions.
Subcellular fractionation location
Separation of the nuclear and cytosolic fractions was performed using the PARIS Kit (Cat. AM1921, Invitrogen, CA, USA) according to the manufacturer’s instructions.
In vitro transcription assays and RNA pull-down mass spectrometry (LC-MS/MS) assays
In vitro translation assays were performed using mMESSAGE mMACHINE™ T7 Transcription Kit according to the manufacturer’s instructions (Cat. AM1344, Invitrogen, CA, USA). Then, HOXC-AS3 RNAs were labeled with desthiobiotinylation using the Pierce RNA 3′ End Desthiobiotinylation Kit (Cat. 20164, Magnetic RNA-Protein Pull-Down Kit, Components, Thermo). RNA pull-down assays were performed with Pierce Magnetic RNA-Protein Pull-Down Kit according to the manufacturer’s instructions (Cat. 20164, Magnetic RNA-Protein Pull-Down Kit, Thermo). After elution of lncRNA-interacting proteins, they were subjected to mass spectrometric analysis. LC-MS/MS experiments were performed with an LTQ linear ion trap mass spectrometer (Thermo Finnigan, San Jose, CA) equipped with a microspray source.
RNA immunoprecipitation (RIP) assays
RNA immunoprecipitation (RIP) experiments were performed using a Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Cat. 17-701, Millipore, USA) according to the manufacturer’s instructions. The antibodies for RIP assays of YBX1 (Cat. ab12148, ab76149) were from Abcam.
Total RNA from BGC-823 cells with HOXC-AS3/YBX1 knockdown and control cells were isolated and quantified. The concentration of each sample was measured with a NanoDrop 2000 (Thermo Scientific, USA). The quality was assessed by an Agilent2200 (Agilent, USA). The sequencing library of each RNA sample was prepared using the Ion Proton Total RNA-Seq Kit v2 according to the protocol provided by the manufacturer (Life Technologies, USA). Data are available in Additional file 5: Table S4 and Additional file 6: Table S5.
Chromatin immunoprecipitation (ChIP) assays
ChIP assays were performed using the EZ-CHIP KIT according to the manufacturer’s instruction (Cat. 17-408, Millipore, USA). The antibodies for Histone H3, acetyl-histone H3 Lys27 (H3K27Ac, Cat. ab4729), and H3 trimethyl Lys4 (H3K4me3, Cat. ab8580) were from Abcam. The ChIP primer sequences were listed in Additional file 7: Table S6. The antibody for YBX1 (Cat. ab12148) was from Abcam. Quantification of immunoprecipitated DNA was performed using qPCR. ChIP data was calculated as a percentage relative to the input DNA from the equation 2[Input Ct − Target Ct] × 100 (%).
All statistical analyses were performed using SPSS 20.0 software (IBM, SPSS, USA). The significance of differences between groups was estimated by Student’s t test, χ2 test, or Wilcoxon test, as appropriate. OS rates were calculated by the Kaplan–Meier method with the log-rank test for comparison. Survival data were evaluated using univariate and multivariate Cox proportional hazards model. Variables with a value of P < 0.05 in univariate analysis were used in the subsequent multivariate analysis based on the Cox regression analyses. Two-sided P values were calculated, and a probability of 0.05 was selected for statistical significance.
Additional methods are described in Additional file 8: Supplementary Methods.
This work was supported by National Natural Science Foundation of China (81702266, 81502071, 81401873, and 81772479), China Postdoctoral Science Foundation (2017M610339 and 2017M611913) and Jiangsu Planned Projects for Postdoctoral Research Funds (1701041A). This work was also supported by Key Project and supported by Medical Science and Technology Development Foundation, Nanjing Department of Health (YKK15145), Foundation of Jiangsu Province Medical Youth Talent (QNRC2016057 and QNRC2016380), and Foundation of Xuzhou Central Hospital (XZB201616). This work was supported by the Scientific Foundation of Wuxi City of Jiangsu (Q201728).
Availability of data and materials
Our RNA-seq data used in this study (RNA-seq after knockdown HOXC-AS3 and YBX1) have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO accession number GSE119021 . The lncRNA expression profiles data were obtained from GEO, with accession numbers GSE50710  and GSE58828 .
EBZ, DDY, LH, WD, and JFC contributed to the conception and design. EBZ, JS, and CGZ contributed to the development of the methodology. DDY, JS, and XYL contributed to the acquisition of data. EBZ and XZH contributed to the writing the manuscript. XXS and XZH contributed to the administrative, technical, and material support. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The study was approved by the Medical Ethical Committee of Southeast University Medical College, and it was performed in compliance with the Helsinki Declaration. All patients have given written informed consent for publication.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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