Background
Recent evidence indicates that although more than 70% of the eukaryotic genome is transcribed, only approximately 1 to 2% of the transcriptome contributes to protein-coding RNA, suggesting that transcription is not limited to the protein-coding portion of the eukaryotic genome but includes other non-protein-coding sections [
1,
2]. Based on their sizes, these transcribed noncoding RNAs (ncRNAs) can be classified as small, medium and long. Small ncRNAs range from 18 to 31 bp in length, whereas long ncRNAs range in size from 200 bp to over several hundred kilobases. Medium ncRNAs measure between 31 bp and 200 bp and contain mainly snRNAs and snoRNAs. Long noncoding RNAs (lncRNAs) are of interest because emerging evidence indicates that a subset of long noncoding RNAs mediate their biological functions using chromatin as a substrate to interact with the genetic information encoded in the genome [
3].
Gastric cancer (GC) is ranked the fifth most common malignant neoplasm in the world, with approximately 951,600 new diagnoses and 723,100 deaths in 2012 [
4]. Despite the decreased mortality rate of GC in recent years, it is still the second most common cause of cancer death [
5,
6]. Further exploration of the molecular mechanisms underlying GC occurrence and development is urgently needed. Studying the aberrantly expressed lncRNAs involved in signaling pathways in GC may provide us with new insights into the occurrence and development of this disease. By acting as oncogenes or tumor suppressors, lncRNAs contribute to GC occurrence and development. Several lncRNAs, such as HULC, MALAT1, lncRNA-ATB and HOTTIP, have been demonstrated oncogenic activity [
7‐
11], while other lncRNAs, including HOTAIR, GAS5 and PTENP1, are considered tumor suppressors [
12‐
14]. The cellular localization of lncRNAs is varied. Majority of lncRNAs are localized to the nucleus (MALAT1 and NEAT1), some are distinctively found in the cytoplasm (DANCR and OIP5-AS1), and certain lncRNAs are found in both locations (TUG1, CasC7 and HOTAIR) [
15]. The cellular localization of lncRNAs is intended for indulging in range of physiological activities from chromatin remodeling to translational regulation [
16]. The basic structural and interactive capabilities of lncRNAs with other cellular biomolecules can help distinguish and specifically reveal their central roles in tumorigenesis. LncRNA–DNA, lncRNA–RNA and lncRNA–protein interactions are especially important. lncRNAs are involved in various levels of regulation, including transcriptional repression by binding to the PRC2 (Polycomb Repressive Complex 2) [
12]. LncRNAs can also serve as a ‘sponge’ to titrate miRNAs, thus participating in post-transcriptional processing [
17,
18]. The most important biomolecular interactions of lncRNAs are with RNA-binding proteins. All the classic molecular mechanisms of lncRNAs, such as guiding, scaffolding and decoying, are ultimately executed through interactions with proteins [
19,
20].
The mammalian target of rapamycin (mTOR) signaling pathway integrates both intracellular and extracellular signals and functions as a central regulator of cell metabolism, growth, proliferation and survival. Activation of the mTOR pathway has a substantial regulatory role in cell proliferation and cell cycle progression [
21]. The mTOR protein forms at least two distinct multiprotein structures: mTOR complex 1 (mTORC1) and 2 (mTORC2). These complexes share the catalytic mTOR subunit, mammalian lethal with sec-13 protein 8 (mLST8, also known as GbL), the DEP domain containing mTOR-interacting protein (DEPTOR), and the Tti1/Tel2 complex. Regulatory-associated protein of mammalian target of rapamycin (raptor) and proline-rich Akt substrate 40 kDa (PRAS40) are specific to the mTORC1 complex, whereas rapamycin-insensitive companion of mTOR (rictor), mammalian stress-activated map kinase-interacting protein 1 (mSin1) and protein observed with rictor 1 and 2 (protor1/2) are only part of the mTORC2 complex [
21].
Here, we report the characterization of HAGLROS as an lncRNA highly expressed in GC and implicated in the regulation of cell proliferation and migration. In the present study, we identified HAGLROS by analyzing publicly available lncRNA expression profiling data from GC. HAGLROS has only one transcript, a 699 bp lncRNA, according the NCBI (NCBI Reference Sequence: NR_110457.1). HAGLROS was up-regulated in GC tissues and served as an independent predictor for overall survival. In addition, HAGLROS was a direct transcriptional target of STAT3, and HAGLROS regulated GC cell proliferation both in vitro and in vivo. Mechanistic studies showed that HAGLROS regulated mTOR signaling by functioning as a competing endogenous RNA (ceRNA), which suppressed the degradation of mTOR mRNA by competing with miR-100-5p. HAGLROS also functioned as an mTORC1 binding partner, interacting with mTOR, Raptor and PRAS40 and stabilizing the complex. Activated mTOR promoted excessive proliferation and maintained the malignant phenotype of GC cells by inhibiting autophagy. Taken together, our study revealed that STAT3-induced lncRNA HAGLROS overexpression contributes to the malignant proliferation and invasion of GC cells via mTOR signal-mediated inhibition of autophagy and predicts poor outcomes in GC patients. These results provide important experimental evidence for the diagnosis and treatment of GC and suggest that HAGLROS may serve as a target for new therapies in human GC.
Methods
Microarray analysis
GC gene expression data were downloaded from The Cancer Genome Atlas (TCGA) and National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) dataset. Heat maps representing differentially regulated genes were generated using Cluster 3.0 software (
http://hemi.biocuckoo.org). Microarray data have been deposited under accession number GSE58828.
Cell lines
The human gastric cancer cell lines SGC-7901, BGC-823, HGC-27, MGC-803 and AGS and the normal gastric epithelium cell line (GES1) were obtained from the Chinese Academy of Sciences Committee on Type Culture Collection Cell Bank (Shanghai, China). They are cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere containing 5% CO2 at 37 °C.
Study subjects
We obtained 84 paired GC and adjacent non-cancerous tissues from patients who underwent surgery at Nanjing First Hospital of Jiangsu Province in China between 2011 and 2012 and who were diagnosed with GC based on histopathologic evaluation. No local or systemic treatment was conducted in these patients before surgery. All collected tissue samples were preserved in RNA Transport (OMEGA Engineering Inc., Norwalk, CT, USA) and immediately frozen at −80 °C until required. The clinical characteristics of all patients are listed in Additional file
1: Table S1.
Antibodies and reagents
The following antibodies were used in the study: mTOR Pathway Antibody Sampler Kit, anti-P70S6K, anti-p-P70S6K, anti-LC3, anti-P62 and secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). 3-MA was purchased from Sigma-Aldrich (St. Louis, MO, USA).
qRT-PCR analysis
qRT-PCR was used to detect expression levels of HAGLROS and other genes in GC tissues and cells following the manufacturer’s instructions (LightCycler® 480, Roche, Basel, Switzerland). GAPDH and β-actin were used as controls. Essential details referred to the MIQE guidelines [
22]. Primers are listed in Additional file
2: Table S2. Total RNA was extracted using Trizol (Invitrogen, Grand Island, NY, USA), according the manufacturer’s instructions. cDNA was synthesized using PrimeScript™ RT reagent Kit (Takara Bio USA, Inc., Mountain View, CA, USA, No. RR047A).
For miRNA quantification, the Bulge-loop™ miRNA qRT-PCR Primer Sets (one RT primer and a pair of qRT-PCR primers for each set) specific for miR-100-5p is designed by RiboBio (Guangzhou, China). cDNA was synthesized using PrimeScript™ RT reagent Kit (Takara Bio USA, No. RR037A).
Plasmid construction and cell transfection
The full-length complementary cDNA of human HAGLROS was synthesized by Invitrogen and cloned into the expression vector pc-DNA3.1 (Takara Bio USA, Inc.) the small hairpin RNA (shRNA) of the HAGLROS was provided by Invitrogen Corporation (Grand Island, NY, USA), and the final construct was verified by sequencing. Plasmid vectors for transfection were prepared using DNA Midiprep Kits (Qiagen, Hilden, Germany) and transfected into GC cells using Lipofectamine 2000 (Invitrogen). The siRNAs were transfected into GC cells using Lipofectamine 2000 according the manufacturer’s instructions. All siRNA and shRNA sequences are listed in Additional file
2: Table S2.
Luciferase reporter assays
The STAT3-binding motif in the promoter region of HAGLROS was identified by JASPAR (
http://jaspar.genereg.net/). The different fragment sequences were synthesized and then inserted into the pGL3-basic vector (OMEGA Engineering Inc.) and co-transfected with STAT3 plasmid into 293T cells. The miR-100-5p sequence was synthesized, inserted into the pGL3-basic vector and co-transfected with wild-type and mutant HAGLROS (the binding site for miR-100-5p was mutated) plasmid into 293T cells. The 3′-UTR of mTOR was cloned into the luciferase vector and transfected into 293T together with miR-100-5p mimics, the miR-100-5p inhibitor, the HAGLROS plasmid or the negative control. All vectors were verified by sequencing, and luciferase activities were assessed using a Dual Luciferase Assay Kit (OMEGA Engineering Inc.) in accordance with the manufacturer’s instructions.
Cell proliferation
Cell proliferation ability was examined using a Cell Proliferation Reagent Kit I (MTT, Sigma-Aldrich). Absorbance values were measured at the wavelength of 490 nm. Inhibitory rates were calculated by Microsoft Excel (Microsoft Corporation, Redmond, WA, USA).
Colony formation assays were performed to monitor the cloning capability of GC cells. Cells were seeded in 6-well plates at 1 × 103 cells/well and cultivated in DMEM (without any cytokine) with 10%FBS for 14 days, with the medium being replaced every 4 days. Colonies were fixed with methanol and stained with 0.1% crystal violet (Sigma-Aldrich) in PBS for 15 min. Colony formation was determined by counting the number of stained colonies.
Cell migration and invasion
A wound healing assay was used to test for cell migration capabilities. A total of 2–4 × 105 cells were seeded in 6-well plates, cultured for 12–24 h, and transfected with siRNAs or a control siRNA and with pc-DNA3.1-HAGLROS or a control vector. Once cultures reached 85% confluency, the cell layer was scratched with a sterile plastic tip, washed with culture medium, and then cultured for 24 h and 48 h. At different time points, images of the plates were acquired using a microscope (Olympus, Tokyo, Japan) and relative areas of wounds using Image J software to quantify and calculate the significance of the observed event.
For the invasion assays, 1 × 105 cells in serum-free medium were placed into the upper chamber of an insert coated with Matrigel. Medium containing 10% FBS was added to the lower chamber. After incubation for 24 h, the cells remaining on the upper membrane were removed with cotton wool. Cells that had migrated or invaded through the membrane were fixed with methanol, stained with 0.1% crystal violet, imaged, and counted using an inverted microscope (Olympus).
Establishment of xenografts and in vivo studies
Animal studies were performed in accordance with the criteria outlined in the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the National Academy of Sciences and published by the National Institutes of Health (USA). Four-week-old female athymic BALB/c nude mice were maintained under specific pathogen-free conditions and manipulated according protocols approved by the Shanghai Medical Experimental Animal Care Commission and the Committee on the Ethics of Animal Experiments of the Nanjing Medical University. HAGLROS shRNA and Ctrl shRNA stably transfected SGC-7901 cells were harvested, and 1 × 107 cells were subcutaneously injected into a single side of each mouse. Tumor sizes were measured by caliper and recorded every 3 days. The tumor volumes were calculated from the length (the longest diameter across the tumor) and width (the corresponding perpendicular diameter) using the following formula: π/6 × length × width2. After 20 days of growth, animals were killed, and tumors were resected and preserved at −80 °C or in formaldehyde for qRT-PCR and IHC staining, respectively.
FISH and subcellular fractionation
FISH assay was performed using a Ribo™ Fluorescent In Situ Hybridization Kit and Ribo™ lncRNA FISH Probe Mix (Ribo, Guangzhou, China) according to the manufacturer’s protocols. The separation of nuclear and cytosolic fractions was performed using a PARIS Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions.
RNA immunoprecipitation
An EZMagna RNA immunoprecipitation (RIP) Kit (Millipore, Bedford, MA, USA) was used following the manufacturer’s protocol. BGC-823 and SGC-7901 cells were lysed in complete RIP lysis buffer (containing proteinase inhibitor and phosphatase inhibitor), and the cell extract was incubated with magnetic beads conjugated with specific antibodies or control IgG for 6 h at 4 °C. Beads were washed and incubated with proteinase K to remove proteins. Finally, purified RNA was subjected to qRT-PCR analysis.
RNA pull-down assay
RNAs were in vitro transcribed using T7 RNA polymerase (Ambion Inc., Austin, TX, USA), purified using an RNeasy Plus Mini Kit (Qiagen), and treated with RNase-free DNase I (Qiagen). Transcribed RNAs were biotin labeled with Biotin RNA Labeling Mix (Ambion Inc.). Positive, negative, and biotinylated RNAs were mixed and incubated with BGC-823 cell lysates. Magnetic beads were added to each binding reaction, followed by incubation at room temperature. Beads were washed with washing buffer, and eluted proteins were examined by Coomassie brilliant blue (Beyotime, Shanghai, China) staining and Western blot analysis.
Chromatin immunoprecipitation
Chromatin immunoprecipitation (ChIP) experiments were performed using the MagnaChIP Kit (Millipore) according to the manufacturer’s instructions as described previously [
23]. ChIP assay related primers are listed in Additional file
2: Table S2.
Western blot analysis and immunoprecipitation
Western blot analysis and immunoprecipitation were performed according to standard protocols as described previously [
24].
LC3-II punctuation assay
For detecting the LC3-II punctuation, BGC-823 cells were transiently co-transfected with GFP-LC3 and HAGLROS siRNAs for 24 h and then seeded in a 24-well plate covered with 14 × 14 mm slips for next 24 h. After that, cells were fixed, permeabilized, and incubated with DAPI for 10 mins, and then adhered to coverslips after PBS washing. Cells on coverslips were observed using a confocal microscope (Olympus).
Immunohistochemical (IHC) analysis
To quantify Ki67 expression, both the intensity and extent of immunoreactivity were evaluated and scored. IHC staining and score evaluation were performed according to standard protocols as described previously [
25].
Statistical analysis
Differences between groups were assessed by a paired, two tailed Student’s t-test. The Chi-square test was used to analyze the pathologic features of HAGLROS expression in GC. The survival curves are drawn using Kaplan-Meier survival plots and tested using log-rank tests. Univariate and multivariate Cox proportional hazards modeling was used to determine the effects of variables on survival. All statistical analyses were performed using SPSS 22.0 software (IBM SPSS, Chicago, IL, USA).
Discussion
Most cancers are genetic diseases that change the flow of cellular information and thus disrupt cellular homeostasis and promote proliferation. More and more evidence confirms that lncRNAs have important roles in GC carcinogenesis, proliferation and metastasis through survival signaling pathways [
29]. Differential display analysis of human prostate cancers identified Differential display 3, also known as PCA3, this lncRNA was approved by the Food and Drug Administration (FDA) for prostate cancer diagnosis and was the first case of a lncRNA being used for clinical testing by FDA approval [
30,
31]. Similarly, analyses of gastric secretions from patients with GC identified lncRNA-AA174084 as a biomarker capable of differentiating between GC and benign disorders of the gastric epithelium [
32]. Many lncRNAs have been reported to be associated with GC, but the most characteristic biomarker remains unclear. In the present study, we found that the lncRNA HAGLROS was significantly overexpressed in GC compared to corresponding non-tumor tissues. The high expression levels of HAGLROS in GC were positively correlated with invasion depth and TNM stage. In addition, high HAGLROS expression in GC tissues was associated with a poor prognosis and could be an independent prognostic indicator. These results suggested that HAGLROS might have important roles in GC progression.
The dysregulation of lncRNAs influences various pathological processes. Alike protein-coding transcripts, the transcription of lncRNAs is subject to typical epigenetic-mediated and transcription factor-mediated regulation. STAT3 is both a transcriptional activator and an oncogene under normal physiological conditions. However, much evidence indicates that STAT3 is constitutively activated in cancers, playing a crucial role in tumor onset and progression. In addition to its traditional role in cancer cell proliferation, invasion, and migration, STAT3, as a transcription factor, promotes cancer development by altering the expression of other genes in cancer cells [
33]. Furthermore, overexpression of STAT3 has been observed in various types of tumors, including GC. In this study, we found that HAGLROS was a direct target of transcription factor STAT3, which was affirmed by STAT3 binding to the predicted site of the promoter region of HAGLROS and by STAT3 causing significant induction of HAGLROS promoter activity, as determined by luciferase reporter assay (Fig.
4). Therefore, up-regulation of HAGLROS in GC is partly due to STAT3 activation during tumor progression.
lncRNA regulation of cellular processes depends in part on lncRNA cellular localization: nuclear lncRNAs are enriched for functionality involving chromatin interactions, transcriptional regulation, and RNA processing, while cytoplasmic lncRNAs can modulate mRNA stability or translation and influence cellular signaling cascades [
1]. LncRNA modulation of RNA metabolism is an emerging theme for lncRNAs that are enriched in the cytoplasm, where the lncRNAs participate in cellular biological processes by functioning as ceRNAs or “RNA sponges” regulating mRNA stability, mRNA alternative splicing, and protein localization [
34]. In the present study, we report that HAGLROS is localized preferentially in the cytoplasm, as determined by subcellular fractionation and FISH experiments. HAGLROS functions as a ceRNA to antagonize miR-100-5p-mediated mTOR mRNA degradation; HAGLROS and mTOR interact through competition for miR-100-5p binding. In addition, we observed that HAGLROS, as a mainly cytoplasmic lncRNA, interacts directly with mTORC1 components (mTOR, Raptor and PRAS40) and activated the mTORC1 pathway by stabilizing the complex’ structure. In accordance with the above statements, cytoplasmic lncRNA HAGLROS has two mechanisms to activate the mTOR pathway and thus inhibit autophagy. On the one hand, HAGLROS functions as a ceRNA to increase mTOR mRNA expression through competing with miR-100-5p, on the other hand, HAGLROS binds mTORC1 key proteins to activate the complex and finally participates in cellular biological processes. mTORC1, a master positive regulator of cell growth and proliferation, forms the integrational hub of an extensive network of regulatory proteins that transmit extrinsic and intrinsic signals regarding cellular nutritional status. The influence of mTORC1 on cellular metabolism is substantial considering its regulation by common oncogenic signaling pathways (e.g., PIK3CA-AKT1 and RAS-ERK) [
35] and the observation that aberrant mTORC1 signaling is found in 40% to 90% of human cancers [
36].
Given the intimate relationship between mTORC1 signaling and autophagy, it is likely that cancer-associated sequence changes in the mechanistic target of rapamycin or mTOR and/or aberrant mTOR protein expression would perturb autophagy, making autophagy an important mediator of the effects of this common dysregulation in human cancer [
37]. Here, we monitored the knockdown of HAGLROS and its effects on autophagy through the mTOR pathway. The functional relevance of autophagy in tumor formation and progression remains controversial. Intriguingly, many oncogenes and tumor suppressor genes affect autophagic pathways, and the dysregulation of the autophagic process contributes to malignant transformation [
38]. Many tumor suppressor proteins, such as p53, phosphatase and tensin homolog (PTEN) and death-associated protein kinase (DAPK), that provide constitutive input signals to activate autophagy are mutated in multiple cancers. Conversely, oncogenes, including Akt, mTOR and Bcl-2, inhibit autophagic processes indicating that elevated autophagy signaling may contribute to tumor suppression [
39,
40]. In GC cells, HAGLROS and mTOR levels are consistently parallel, with increased levels inhibiting autophagy and promoting tumor proliferation and invasion. The regulatory mechanism of HAGLROS was summarized in the Fig.
8f.