Background
Gastric cancer is the second leading cause of cancer-induced death, and is the most common gastrointestinal malignancy in East Asia, Eastern Europe, and parts of Central and South America. In most patients, gastric cancer is diagnosed at an advanced stage and is accompanied by malignant proliferation, extensive invasion and lymphatic metastasis. Successful therapeutic strategies are limited and the mortality is high [
1,
2]. Long non-coding RNAs (lncRNAs) have recently gained significant attention in delineating the complex mechanisms underlying malignant processes such as carcinogenesis, metastasis and drug resistance. Therefore, if we want to fully understand gastric carcinogenesis, we need to consider this family of regulatory transcripts that add a new layer of complexity to tumor biology.
Although only a small number of functional lncRNAs have been well characterized to date, they have been shown to regulate gene expression at various levels, including chromatin modification, transcription and post-transcriptional processing [
3,
4]. Recently, a new regulatory mechanism has been identified in which crosstalk between lncRNAs and mRNAs occurs by competing for shared microRNAs (miRNAs) response elements. In this case, lncRNAs may function as competing endogenous RNAs (ceRNAs) to sponge miRNAs, thereby modulating the derepression of miRNA targets and imposing an additional level of post-transcriptional regulation [
5]. In previous reports, a muscle-specific lncRNA, linc-MD1, has been reported to be a ceRNA that protects MyoD messenger RNA (mRNA) from miRNA-mediated degradation [
6]. Pluripotency-associated lnc-RoR may function as a key ceRNA to link the network of miRNAs and core transcription factors, e.g., Oct4, Sox2, and Nanog, in human embryonic stem cells. Notably, lncRNA HULC is highly upregulated in liver cancer and plays an important role in tumorigenesis [
7]. In particular, HULC may act as an endogenous ‘sponge’ that down-regulates a series of miRNAs activities, including miR-372 [
8]. We therefore propose that some lncRNAs may also have roles as ceRNAs, linking miRNAs and the post-transcriptional network in gastric pathogenesis.
HOTAIR (
Hox transcript antisense intergenic RNA) is a ~2.2-kb long non-coding RNA transcribed from the
HOXC locus, which can repress transcription
in trans of
HOXD in foreskin fibroblasts [
9]. As a novel molecule in the field of tumor biology, HOTAIR initially became well known for its involvement in primary breast tumors and breast cancer metastases, wherein elevation of HOTAIR promoted invasiveness and metastasis [
10]. Furthermore, HOTAIR expression positively correlates with malignant processes and poor outcome in colorectal cancer, hepatocellular carcinoma, pancreatic cancer and gastrointestinal stromal tumors [
11‐
14]. Recent studies reported that HOTAIR was upregulated in gastric cancer [
15,
16]. Nevertheless, the overall biological role and underlying molecular mechanism of HOTAIR in gastric carcinogenesis remains largely undefined.
In this study, we report that HOTAIR upregulation is a characteristic molecular change in gastric cancer and investigate the biological roles of HOTAIR on the phenotypes of gastric cancer cells in vitro and in vivo. Moreover, mechanistic analysis reveals that HOTAIR may function as a ceRNA to regulate the expression of human epithelial growth factor receptor 2 (HER2) through competition for miR-331-3p, thus playing an oncogenic role in gastric pathogenesis. The present work provides the first evidence for a positive HOTAIR/HER2 correlation and the crosstalk between miR-331-3p, HOTAIR and HER2, shedding new light on the treatment of gastric cancer.
Discussion
LncRNAs, which are more than 200 nucleotides in length with limited protein-coding capacity, are often expressed in a disease-, tissue- or developmental stage-specific manner, indicating specific functions for lncRNAs in development and diseases and making these molecules attractive therapeutic targets [
20‐
22]. A number of recent papers have revealed that dysregulation of these lncRNAs may also affect the regulation of the eukaryotic genome and provide a cellular growth advantage, resulting in progressive and uncontrolled tumor growth [
23‐
25]. Therefore, lncRNAs may provide a missing piece of the otherwise well-known oncogenic and tumor suppressor network puzzle.
In this study, we tested the expression of HOTAIR in gastric carcinoma samples and their surrounding non-tumorous tissues. We also identified the function of HOTAIR in gastric carcinoma cells by applying gain- and loss-of-function approaches. The results demonstrated that HOTAIR was upregulated in gastric carcinoma tissues in comparison with adjacent normal gastric tissues, and that HOTAIR upregulation correlated with larger tumor size, advanced pathological stage and extensive metastasis. Moreover, the overall survival time of patients with lower HOTAIR expression levels was significantly longer than that of patients with moderate or strong HOTAIR expression levels. Furthermore, HOTAIR overexpression promoted the proliferation, migration and invasion of gastric carcinoma cells, while HOTAIR depletion inhibited cell invasion and cell viability, and induced growth arrest both in vitro and in vivo. Additionally, HOTAIR suppression led to the promotion of gastric cell apoptosis. These findings suggest that HOTAIR plays a direct role in the modulation of multiple oncogenic properties and gastric cancer progression, stimulating new research directions and therapeutic options considering HOTAIR as a novel prognostic marker and therapeutic target in gastric cancer.
The importance of lncRNAs in human disease may be associated with their ability to impact cellular functions through various mechanisms. In this study, as far as the mechanism of HOTAIR is concerned, it is worth mentioning that subcellular localization analysis of HOTAIR by RNA fluorescence in situ hybridization assay demonstrates the localization of HOTAIR to both the nucleus and the cytoplasm [
24]. It is evident that nuclear HOTAIR can target polycomb repressive complex 2, altering H3K27 methylation and gene expression patterns across the genome [
10,
11]. Recent work reported a scaffold function for HOTAIR in the cytoplasm as an inducer of ubiquitin-mediated proteolysis [
26]. Nevertheless, the tumorigenic properties and mechanistic heterogeneity of HOTAIR, and particularly those of the cytoplasmic form, are far from being fully elucidated.
Inspired by the ‘competitive endogenous RNAs’ regulatory network and emerging evidence that suggests that lncRNAs may participate in this regulatory circuitry, we hypothesized that HOTAIR may also serve as a ceRNA and so we searched for potential interactions with miRNAs. In support of this notion, we employed bioinformatics analysis and luciferase assays to validate the direct binding ability of the predicted miRNA response elements on the full-length HOTAIR transcript. As expected, we discovered miR-331-3p and miR-124 could form complementary base pairing with HOTAIR and induce translational repression of a RLuc-HOTAIR reporter gene. In addition, HOTAIR:miR-331-3P coimmunoprecipitation with anti-Ago2 demonstrated a physical interaction in gastric cancer cells, providing further support for HOTAIR’s miRNA-sequestering activity. To serve as an endogenous ‘sponge’, the abundance of HOTAIR should be comparable to or higher than miR-331-3p/miR-124. In our study, qRT-PCR analysis showed that miR-331-3p/miR-124 expression was inversely correlated with HOTAIR expression in advanced gastric cancer. Moreover, ectopic overexpression of miR-331-3p or miR-124 expression could arrest gastric cancer proliferation, which was consistent with results of knockdown of HOTAIR expression in gastric cancer cells. Taken together, these data are consistent with our hypothesis and indicate that HOTAIR may interact with miRNAs to link miRNAs and the post-transcriptional network in gastric pathogenesis.
To investigate the miRNA-related functions of HOTAIR in gastric pathogenesis, we chose miR-331-3p as a model miRNA for further studies, with a particular focus on the target gene HER2. In carcinomas, HER2 acts as an oncogene, encoding a 185-kDa transmembrane protein to trigger the activation of cell signaling networks, impacting on various malignant cell functions such as proliferation, motility, angiogenesis and apoptosis [
27‐
29]. HER2 amplification and/or overexpression have been detected in approximately 20% to 30% of patients with breast and gastric cancer and correlates with poorer clinical outcomes [
19,
30,
31]. The importance of HER2 has been well documented in breast cancer, where HER2 testing is a standard approach for identifying patients who may benefit from HER2-targeted agents such as lapatinib and trastuzumab therapy in metastatic and adjuvant settings [
32,
33]. In gastric cancer, HER2 overexpression is associated with more aggressive disease and poor survival. Preclinical studies have indicated that trastuzumab can impede HER2-overexpressing human gastric cancer cells growth and inhibit tumorigenesis in xenograft models [
34‐
36]. Accumulating studies indicate that HER2 overexpression may not be affected by gene amplification alone, but is also likely to be influenced by transcriptional activation and/or post-transcriptional mechanisms in cancers [
28,
37]. In previous reports, HER2 mRNA and protein overexpression have been directly affected by miRNA-mediated post-transcriptional mechanisms in carcinomas [
38,
39]. Our study also confirms that HER2 is a direct target of miR-331-3p. Considering the interaction of HOTAIR/miR-331-3p, we therefore hypothesize that HOTAIR may also regulate HER2 expression in gastric cancer, which signifies the role of HOTAIR in the tumorigenesis-regulating network.
In this study, luciferase and RIP assays confirmed the existence of specific crosstalk between the lncRNA HOTAIR and HER2 mRNA through competition for miR-331-3p binding. Consistent with HOTAIR sequestration of miR-331-3p, we found that its depletion reduced the expression level of HER2, while its overexpression restored elevated HER2 protein synthesis. These data are consistent with the hypothesis that ceRNAs are transmodulators of gene expression through competing miRNA binding. Furthermore, IHC and qRT-PCR assays revealed that HER2 was mainly upregulated in advanced stage gastric cancer tissues or those with lymph node metastasis, and associated with high HOTAIR expression. Altogether, the positive correlation between HOTAIR and HER2 expression and the relevance to miRNA expression levels (miR-331-3p/miR-124) supports our hypothesis that ceRNA can sequester miRNAs, thereby protecting their target RNAs from repression.
Lastly, the findings presented in this study have allowed us to conclude that HOTAIR overexpression represents an excellent biomarker of poor prognosis in gastric cancer, and may confer multiple properties required for tumor progression and metastatic phenotype. More importantly, our study indicates that the ceRNA activity of HOTAIR imparts a miRNA/lncRNA trans-regulatory function to protein-coding mRNAs and the ceRNA network may play an important role in gastric pathogenesis. Finally, our experimental data suggest that targeting the HOTAIR/HER2 interaction may represent a novel therapeutic application, thus contributing to better knowledge of the efficacy and tolerance of trastuzumab-based therapy in HER2-positive gastric cancer patients.
It is worth mentioning that the ceRNA activity of HOTAIR may sequester a handful of miRNAs at once, while one miRNA is also capable of controlling multiple genes. Therefore, the multiple properties of HOTAIR are likely due to simultaneous targeting of multiple targets in gastric cancer. We also hypothesize that there may be many other lncRNAs that function as ceRNAs to regulate expression of key genes in gastric cancer. Thus, the identification of these ceRNAs will undoubtedly enhance our knowledge of how lncRNAs function, allowing us to better understand the pathogenesis and development of gastric cancer and ultimately facilitate the development of lncRNA-directed diagnostics and therapeutics against this deadly disease.
Materials and methods
Tissue collection
Fresh-frozen and paraffin-embedded gastric cancer tissues and corresponding adjacent non-tumorous gastric samples were obtained from Chinese patients at Jiangsu province hospital between 2006 and 2008. All cases were reviewed by pathologist and histologically confirmed as gastric cancer (stageII,III,IV; 7th Edition AJCC) based on histopathological evaluation. Clinical pathology information was available for all samples (Table
1). No local or systemic treatment was conducted in these patients before the operation. The study was approved by the Research Ethics Committee of Nanjing Medical University, China. Informed consents were obtained from all patients.
Cell lines and culture conditions
Four gastric cancer cell lines (MGC-803, SGC-7901, BGC-823, and AGS), a normal gastric epithelium cell line (GES-1), a NSCLC cell line (SPC-A1), a normal human bronchial epithelial cell line (16HBE), two breast cancer cell lines (MCF-7, MDA-MB-231), and a human embryonic kidney cell line (HEK293T) were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI 1640 or DMEM (GIBCO-BRL) medium supplemented with 10% fetal bovine serum (10% FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin (Invitrogen) in humidified air at 37 °C with 5% CO2.
RNA extraction and qRT-PCR analyses
Total RNA was extracted from tissues or cultured cells using TRIZOL reagent (Invitrogen, Carlsbad, Calif). For qRT-PCR, RNA was reverse transcribed to cDNA by using a Reverse Transcription Kit (Takara, Dalian, China). Real-time PCR analyses were performed with Power SYBR Green (Takara, Dalian China). Results were normalized to the expression of GAPDH. For miR-331-3p and miR-124 expression detection, reverse transcription was performed following Applied Biosystems TaqMan MicroRNA Assay protocol (Cat. # 4427975 and Cat. # 4427975). U6 snoRNA was validated as the normalizer. The primers were listed in Additional file
3: Table S4. qRT-PCR and data collection were performed on ABI 7500.
Plasmid constructs
HOTAIR cDNA was cloned into the mammalian expression vector pcDNA3.1 (Invitrogen). To express miRNAs, human microRNA precursors with about 80 bp of flanking sequences in both sides were amplified and cloned into the modified pLL3.7 vector (Invitrogen). To construct luciferase reporter vectors, HER2 3’-UTR and HOTAIR cDNA fragment containing the predicted potential microRNAs binding sites were amplified by PCR, and then subcloned downstream of the luciferase gene in the pLUC luciferase vector (Ambion, Inc.,Austin, TX, USA). Primers for subcloning and plasmid construction were listed in Additional file
4: Table S3. We also designed shRNA sequence targeted HOTAIR as shown in Additional file
3: Table S4. After annealing of the complementary shRNA oligonucleotides, we ligated the annealed oligonucleotides into pENTR vector (sh-HOTAIR).
Transfection of gastric cancer cells
All plasmid vectors for transfection were extracted by DNA Midiprep kit (Qiagen, Hilden, Germany). Three individual HOTAIR siRNAs (si-HOTAIR) and scrambled negative control siRNA (si-NC) were purchased from Invitrogen (Invitrogen, CA, USA). Target sequences for HOTAIR siRNAs were listed in Additional file
3: Table S4. The si-HOTAIR, miR-331-3p or miR-124 was transfected into BGC-823 cells respectively, and pCDNA/HOTAIR was transfected into SGC-7901 cells using Lipofectamine2000 (Invitrogen) according to the manufacturer’s instructions. At 48 h after transfection, cells were harvested for qRT-PCR analyses or western blot.
Cell proliferation assays
A cell proliferation assay was performed with MTT kit (Sigma, St. Louis, Mo) according to the manufacturer’s instruction. Cells were placed into 6-well plate and maintained in media containing 10% FBS for 2 weeks. Colonies were fixed with methanol and stained with 0.1% crystal violet (Sigma, St. Louis, Mo). Visible colonies were manually counted.
Flow-cytometric analysis of apoptosis
BGC-823 cells transiently transfected with si-NC or si-HOTAIR were harvested 48 h after transfection by trypsinization. After the double staining with FITC-Annexin V and Propidium iodide (PI), the cells were analyzed with a flow cytometry (FACScan®; BD Biosciences) equipped with a CellQuest software (BD Biosciences).
Hoechst staining assay
BGC-823 cells transiently transfected with si-NC or si-HOTAIR were cultured in six-well cell culture plates, and Hoechst 33342 (Sigma, St Louis, MO, USA) was added to the culture medium; changes in nuclear morphology were detected by fluorescence microscopy using a filter for Hoechst 33342 (365 nm). For quantification of Hoechst 33342 staining, the percentage of Hoechst-positive nuclei per optical field (at least 50 fields) was counted.
Cell migration and invasion assays
At 48 h after transfection, cells in serum-free media were placed into the upper chamber of an insert for migration assays (8-μm pore size, millepore) and invasion assays with Matrigel (Sigma-Aldrich, USA). Media containing 10% FBS was added to the lower chamber. After several hours of incubation, the cells that had migrated or invaded through the membrane were stained with methanol and 0.1% crystal violet, imaged, and counted using an IX71 inverted microscope (Olympus, Tokyo, Japan).
5-week-old female athymic BALB/c mice were purchased from the Model Animal Research Center of Nanjing University. All animal procedures were performed in accordance to the protocols approved by the Institutional Animal Care and Use Committee at the Nanjing Medical University. For xenograft models, 5 × 106 BGC-823 cells transfected with sh-HOTAIR and pENTR vector (EV) were injected subcutaneously in the right flank of BALB/c nude mice (five mice per group). Tumor volumes were examined every 3 days when the implantations were starting to grow bigger. After 16 days, these mice were sacrificed and tumors were weighted. Tumor volumes were calculated by using the equation V (mm3) = A × B2/2, where A is the largest diameter, and B is the perpendicular diameter. The primary tumors were excised and tumor tissues were used to perform qRT-PCR analysis of HOTAIR levels and immunostaining analysis of proliferating cell nuclear antigen (PCNA) protein expression.
Luciferase assay
Human HEK293T cells (2.0 × 104) grown in a 96-well plate were co-transfected with 150 ng of either empty vector or miR-331-3p, miR-124, 50 ng of firefly luciferase reporter comprising 3’UTR of HER2, wild type or mutant HOTAIR fragment, and 2 ng of pRL-TK (Promega, Madison, WI, USA) using Lipofectamie 2000 (Invitrogen, USA). rno-miRNA-344 acts as a negative control. Cells were harvested 48 h after transfection for luciferase assay using a luciferase assay kit (Promega) according to the manufacturer’s protocol. Transfection was repeated in triplicate.
RNA Binding Protein Immunoprecipitation (RIP) assay
RNA immunoprecipitation was performed using the EZ-Magna RIP kit (Millipore, Billerica, MA, USA) following the manufacturer’s protocol. BGC-823 cells at 80-90% confluency were scraped off, then lysed in complete RIP lysis buffer, after which 100 μl of whole cell extract was incubated with RIP buffer containing magnetic beads conjugated with human anti-Ago2 antibody (Millipore), negative control normal mouse IgG (Millipore). Anti-SNRNP70 (Millipore)was used as positive control for the RIP procedure. Samples were incubated with Proteinase K with shaking to digest the protein and then immunoprecipitated RNA was isolated. The RNA concentration was measured using a NanoDrop (Thermo Scientific) and the RNA quality assessed using a bioanalyser (Agilent, Santa Clara, CA, USA). Furthermore, purified RNA was subjected to qRT-PCR analysis to demonstrate the presence of the binding targets using respective primers.
Western blot assay and antibodies
Cells protein lysates were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to 0.22 μm NC membranes (Sigma) and incubated with specific antibodies. Autoradiograms were quantified by densitometry (Quantity One software; Bio-Rad). GAPDH antibody was used as control, anti-HER2 (1:1000) and cleaved caspase-3 (1:1,000) were purchased from Cell Signaling Technology, Inc (CST).
Immunohistochemistory (IHC)
Paraffin-embedded, formalin-fixed tissues were immunostained for HER2 and PCNA proteins. The signal was amplified and visualized with diaminobenzidine-chromogen, followed by counterstaining with hematoxylin. For HER2, an IHC score of 2+ or more was defined as positive, and IHC scores of 0 and 1+ were defined as negative [
40]. Anti-HER2 (1:50) was purchased from Cell Signaling Technology, Inc. (CST), and anti-PCNA (1:50) was purchased from Bioworld Technology, Inc., respectively.
Statistical analysis
Student’s t-test (two-tailed), One-way ANOVA and Mann–Whitney test were performed to analyze the in vitro and in vivo data using SPSS 16.0 software. P values less than 0.05 were considered significantly.
Competing interests
All the authors hereby declare that they do not have any competing interests with regard to the manuscript submitted here for review.
Authors’ contributions
XHL, MS, KMW carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. YBG carried out the immunoassays. FQN, EBZ, DDY, RK, KHL, XFC, JHL participated in the design of the study and performed the statistical analysis. WD, ZXW conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.