Long non-coding RNAs (lncRNAs), a recently characterized class of non-coding RNAs, have been shown to have important regulatory roles and are de-regulated in a variety of tumors. However, the contributions of lncRNAs to gastric carcinoma and their functional mechanisms remain largely unknown. In this study, we found that lncRNA gastric carcinoma high expressed transcript 1 (lncRNA-GHET1) was up-regulated in gastric carcinoma. The over-expression of this lncRNA correlates with tumor size, tumor invasion and poor survival. Gain-of-function and loss-of-function analyses demonstrated that GHET1 over-expression promotes the proliferation of gastric carcinoma cells in vitro and in vivo. Knockdown of GHET1 inhibits the proliferation of gastric carcinoma cells. RNA pull-down and immunoprecipitation assays confirmed that GHET1 physically associates with insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) and enhances the physical interaction between c-Myc mRNA and IGF2BP1, consequently increasing the stability of c-Myc mRNA and expression. The expression of GHET1 and c-Myc is strongly correlated in gastric carcinoma tissues. Depletion of c-Myc abolishes the effects of GHET1 on proliferation of gastric carcinoma cells. Taken together, these findings indicate that GHET1 plays a pivotal role in gastric carcinoma cell proliferation via increasing c-Myc mRNA stability and expression, which suggests potential use of GHET1 for the prognosis and treatment of gastric carcinoma.
insulin-like growth factor 2 mRNA binding protein 1
long non-coding RNA
Gastric carcinoma is the fourth most frequent cancer and second most frequent cause of cancer-related deaths worldwide . Despite efforts using diagnostic techniques and patient management, there has been little progress in improving the overall survival of gastric carcinoma patients [2-4]. Development of suitable therapy for the purpose of increasing survival has been limited because the pathophysiological mechanisms contributing to gastric carcinoma are largely unknown [5, 6]. Therefore, revealing the molecular mechanisms for development and progression of gastric carcinoma is vital for developing effective therapies [7, 8].
Recently, studies using a combination of various genome-wide approaches, such as the ENCODE project, have shown that the majority of the mammalian genome is transcribed, but only approximately 1.2% of these transcripts represent protein-coding genes . The large number of non-protein-coding transcripts show cell-specific and dynamic developmental expression patterns, and have structural, regulatory or unknown functions . Among these non-protein-coding transcripts, long non-coding RNAs (lncRNAs), which are greater than 200 nt in length, are characterized by the diversity and complexity of their sequences and action mechanisms . lncRNAs have been shown to play integral roles in control of a wide array of cellular functions, such as cellular growth, apoptosis, division and differentiation [12-14]. Furthermore, several lncRNAs are de-regulated in a variety of disease states, particularly tumors . Perturbation of lncRNA expression may result in aberrant expression of gene products and contribute to the carcinogenesis and progression of tumors . However, there are only preliminary studies of the role of lncRNAs in gastric carcinoma. In a previous study, we found that the lncRNA colon cancer-associated transcript 1 (CCAT1) was up-regulated in gastric carcinoma and activated by c-Myc . However, further investigation is required to determine whether lncRNAs contribute to the prognosis of gastric carcinoma patients, and their functional mechanisms.
In a primary screen of aberrantly expressed lncRNAs in gastric carcinoma, we found that the lncRNA gastric carcinoma high expressed transcript 1 (GHET1, National Center for Biotechnology Information database number AK123072) was highly expressed in the majority of gastric carcinoma tissues examined. In this study, we further analyzed the expression of GHET1 and its association with clinicopathological characteristics and patient prognosis. Our results demonstrate that GHET1 binds insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1), enhances the physical interaction between the IGF2BP1 protein and c-Myc mRNA, up-regulates c-Myc mRNA stability and expression, and promotes the proliferation of gastric carcinoma cells in vitro and in vivo.
GHET1 is up-regulated in gastric carcinoma tissues and is associated with poor prognosis
The expression level of GHET1 was examined using real-time RT-PCR in gastric carcinoma tissue and pair-matched adjacent normal gastric tissues from 42 gastric carcinoma patients. As shown in Fig. 1A, the GHET1 levels were significantly increased in gastric carcinoma tissues compared with adjacent normal gastric tissues (P <0.001 by Wilcoxon signed-rank test).
Next we analyzed the correlation between the GHET1 expression level and the clinicopathological characteristics of the 42 gastric carcinoma patients (Table 1). Correlation regression analysis showed that increased GHET1 expression significantly correlated with tumor size (P =0.011) and invasion (P =0.005).
Table 1. Association between clinicopathological characteristics and GHET1 expression.
The median expression level was used as the cut-off. Low GHET1 expression in the 42 patients was defined as a value below the 50th percentile. High GHET1 expression in the 42 patients was defined as a value above the 50th percentile.
Pearson χ2 tests were used for correlation analysis of the expression levels of GHET1 and clinical features. The results were considered statistically significant at P <0.05.
Tumor size (mm)
Lymph node metastasis
We further determined whether the GHET1 expression level correlated with the outcome for gastric carcinoma patients. Kaplan–Meier survival estimates showed that high GHET1 expression in gastric carcinoma tissues is significantly associated with worse overall survival (P =0.039, log-rank test) (Fig. 1B). These results indicate that GHET1 may play a pivotal role in the pathogenesis of gastric carcinoma and malignant outcomes for patients with gastric carcinoma.
GHET1 promotes the proliferation of gastric carcinoma cells in vitro
Because GHET1 is over-expressed in gastric carcinoma and associated with tumor size, invasion and poor prognosis of gastric carcinoma patients, we next investigated the biological function of GHET1 in gastric carcinoma. We first determined the transcription start and termination sites and full-length sequence of GHET1 using 5′ and 3′ RACE analyses (Fig. S1).
We enhanced GHET1 expression by transfecting a GHET1 expression vector (pcDNA3.1-GHET1) into the gastric carcinoma cell lines MKN45 and AGS, using the pcDNA3.1 vector as a negative control (Fig. 2A). We also inhibited GHET1 expression by transfecting GHET1-specific siRNAs into MKN45 and AGS cells, using control siRNA as a negative control (Fig. 2B). CCK-8 assays indicated that enhanced GHET1 expression promoted cell proliferation in MKN45 and AGS cells (Fig. 2C). In contrast, the inhibition of GHET1 hindered MKN45 and AGS cell proliferation (Fig. 2D).
Furthermore, ethynyl deoxyuridine (EdU) incorporation analysis showed that GHET1-over-expressing MKN45 and AGS cells had higher numbers of EdU-positive nuclei than control cells (Fig. 2E), but the number of EdU-positive nuclei significantly decreased after GHET1 knockdown (Fig. 2F).
The growth-enhancing effect of GHET1 was further demonstrated using colony formation assays. Significantly more colonies were formed by pcDNA3.1-GHET1-transfected MKN45 and AGS cells compared with pcDNA3.1-transfected cells (Fig. 2G). Additionally, depletion of GHET1 expression significantly inhibited the clonogenicity of the MKN45 and AGS cells (Fig. 2H). These results suggest that GHET1 plays an important role in regulating gastric carcinoma cell proliferation.
GHET1 up-regulates c-Myc by increasing c-Myc mRNA stability
As shown in previous studies, c-Myc is regarded as an oncoprotein and is up-regulated in gastric carcinoma [18-21]. Moreover, in our previous study, we found that c-Myc promotes gastric carcinoma cell proliferation . To understand the molecular mechanism by which GHET1 increases gastric carcinoma cell proliferation, we examined whether c-Myc is a downstream GHET1 effector that mediates the function of GHET1 in gastric carcinoma. As shown in Fig. 3A–D, enhanced GHET1 expression significantly increased the mRNA and protein level of c-Myc in MKN45 and AGS cells, while inhibition of GHET1 expression significantly decreased the mRNA and protein level of c-Myc in MKN45 and AGS cells.
Because GHET1 up-regulates the expression of c-Myc, we performed a correlation analysis of the c-Myc mRNA and GHET1 expression levels in the same gastric carcinoma tissues shown in Fig. 1A. A statistically significant correlation was found between c-Myc mRNA and GHET1 (r =0.5512, P =0.0002, Pearson's correlation, Fig. 3E), supporting a role for GHET1 in expression of c-Myc.
To test whether GHET1 regulates the synthesis or degradation of c-Myc mRNA, we induced or inhibited GHET1 expression in MKN45 cells, treated the cells with α-amanitin or dimethylsulfoxide (negative control) 48 h later to block new RNA synthesis, and then measured the loss of c-Myc, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 18S RNA over a 24 h period. Over-expression of GHET1 extended the half-life of c-Myc mRNA, and, conversely, the inhibition of GHET1 shortened the half-life of c-Myc mRNA (Fig. 3F,G). Neither induction of expression nor inhibition of GHET1 altered the half-life of GAPDH mRNA. Collectively, these data demonstrate that GHET1 specifically increases the stability of c-Myc mRNA and up-regulates its expression.
GHET1 associates with IGF2BP1 and enhances the physical interaction between c-Myc mRNA and IGF2BP1 protein
Previous reports have shown that IGF2BP1 physically associates with c-Myc mRNA and prevents c-Myc mRNA degradation [22, 23]. Because IGF2BP1 is an RNA-binding protein, we speculated that lncRNA-GHET1 may also bind IGF2BP1 and influence the association between IGF2BP1 and c-Myc mRNA. To test this hypothesis, we first performed RNA immunoprecipitation (RIP) with an antibody directed against IGF2BP1 using cell extracts from MKN45 cells. We found a significant enrichment of GHET1 mRNA (but not GAPDH mRNA) using the IGF2BP1 antibody compared with the IgG control antibody (Fig. 4A). Consistent with a previous report, c-Myc mRNA enrichment was also observed. To further confirm the association between GHET1 and IGF2BP1, we performed an RNA pull-down experiment. Figure 4B shows a significant enrichment of IGF2BP1 (but not GAPDH protein enrichment) in the presence of GHET1 RNA compared with antisense RNA (negative control). These results demonstrate a specific association between GHET1 and IGF2BP1.
To further test whether GHET1 influences the association between IGF2BP1 and c-Myc mRNA by binding IGF2BP1, we performed RIP experiments with an antibody directed against IGF2BP1 using cell extracts from MKN45 cells transfected with pcDNA3.1 or pcDNA3.1-GHET1, and then measured c-Myc mRNA levels by quantitative RT-PCR. As shown in Fig. 4C, enhanced GHET1 expression significantly increased c-Myc mRNA enrichment as determined using the IGF2BP1 antibody (Fig. 4C). Conversely, inhibition of GHET1 significantly reduced c-Myc mRNA enrichment (Fig. 4D). These data suggest that GHET1 enhances the physical interaction between c-Myc mRNA and IGF2BP1 protein.
To verify whether GHET1 increases c-Myc mRNA stability and up-regulates c-Myc expression through IGF2BP1, we first inhibited IGF2BP1 expression in MKN45 cells (Fig. S2) and transfected pcDNA3.1 or pcDNA3.1-GHET1 into IGF2BP1-inhibited cells 24 h later. As shown in Fig. 4E,F, the c-Myc mRNA and protein level was not changed. These data demonstrate that up-regulation of c-Myc by GHET1 is dependent on IGF2BP1.
c-Myc is critical for GHET1-induced proliferation
Functional assays were used to clarify the importance of c-Myc in enhancement of proliferation by GHET1. c-Myc siRNA and pcDNA3.1 or c-Myc siRNA and pcDNA3.1-GHET1 were co-transfected into MKN45 cells. CCK-8 assays and EdU incorporation analyses indicated that the two groups showed no significant difference in cell proliferation (Fig. 5A,B). In addition, enhanced GHET1 expression did not increase clonogenicity when c-Myc was depleted (Fig. 5C).
GHET1 promotes xenograft tumor growth in nude mice
We examined whether GHET1 influences the growth of gastric cancer cells in nude mice in vivo. MKN45 cells labeled with firefly luciferase and stably transfected with pcDNA3.1-GHET1 or pcDNA3.1 were subcutaneously implanted into the flanks of nude mice. As shown in Fig. 6A–C, the growth of tumors from GHET1-over-expressing xenografts was significantly increased compared with that of tumors from control xenografts.
In this study, we examined expression of the novel long non-coding RNA GHET1 in gastric carcinoma tissues and surrounding normal gastric tissues. We also identified the function of GHET1 in gastric carcinoma cells using gain-of-function and loss-of-function approaches in vitro and in vivo. Our results demonstrate that GHET1 is up-regulated in gastric carcinoma tissues in comparison with adjacent normal gastric tissues, and that GHET1 up-regulation is correlated with tumor size and invasion. High GHET1 expression in gastric carcinoma tissues indicates poor overall survival of gastric carcinoma patients. Enhanced GHET1 expression promotes proliferation of gastric carcinoma cells in vitro and in vivo, while inhibition of GHET1 expression inhibits gastric carcinoma cell proliferation. Therefore, our results indicate that GHET1 functions as an oncogene in gastric carcinoma.
lncRNAs may function to regulate gene transcription by binding promoter regions and/or changing histone markers and chromatin state . In addition, they may interact with and alter the activity of proteins, which may be important for cancer biology [25, 26]. In this study, we found that GHET1, which is located in an intergenic region on chromatin 7, exerts its effect through the above-mentioned mechanism. GHET1 physically associates with IGF2BP1, and enhances the physical interaction between c-Myc mRNA and IGF2BP1 protein. As a result, GHET1 increases c-Myc mRNA stability and expression, consistent with reports that IGF2BP1 physically associates with c-Myc mRNA and prevents c-Myc mRNA degradation . The correlation between c-Myc mRNA and GHET1 expression in clinical gastric carcinoma tissues supports the role of GHET1 in c-Myc stability. Our results also demonstrated that the pro-proliferation role of GHET1 is dependent on c-Myc, and the up-regulation of c-Myc by GHET1 is dependent on IGF2BP1. Other RNA-binding proteins, such as heterogeneous nuclear ribonucleoprotein U (HNRNPU), synaptotagmin binding, cytoplasmic RNA interacting protein (SYNCRIP), Y box binding protein 1 (YBX1) and DEAH (Asp-Glu-Ala-His) box helicase 9 (DHX9), may also associate and cooperate with IGF2BP1 in promoting stabilization of c-Myc mRNA . Whether GHET1 also physically associates with the above-mentioned RNA-binding proteins and/or c-Myc mRNA, and the direct or indirect physical interaction between each component of the specific complexes requires further investigation.
IGF2BP1, which belongs to a conserved family of RNA-binding proteins, is mainly expressed in the embryo and is de novo synthesized in various malignancies . IGF2BP1 associates with various other RNA-binding proteins and forms a distinct ribonucleoprotein complex . By targeting various RNAs, IGF2BP1 has many functions, including modulating cell polarization, proliferation, adhesion and migration [29-31]. In this study, we found that GHET1 further enhanced the effects of IGF2BP1 on c-Myc mRNA stability, but whether GHET1 also influences other functions of IGF2BP1 via association with IGF2BP1 requires further study.
Collectively, our studies indicate that GHET1 is a prognostic factor for gastric carcinoma that promotes c-Myc mRNA stability and expression, and also promotes the growth of gastric carcinoma. These findings indicate that GHET1 is an important molecular marker for determining prognosis, and has the potential to be an important target for gastric carcinoma therapy.
A total of 42 gastric carcinoma tissues and pair-matched adjacent normal gastric tissues were obtained with informed consent from patients who underwent radical resections at Changhai Hospital (Second Military Medical University, Shanghai, China). This study was performed with the approval of the Changhai Hospital Institutional Review Board.
Cell cultures and treatments
The human gastric carcinoma cell lines MKN45 and AGS were obtained from the Chinese Academy of Sciences Cell Bank. The cells were grown in RPMI-1640 with 10% fetal bovine serum (Gibco BRL, Gaithersburg, MD, USA), and maintained in a humidified 37 °C incubator with a 5% CO2 atmosphere. Where indicated, cells were treated with 50 μm α-amanitin (Sigma-Aldrich, St Louis, MO, USA) for the indicated time.
Total RNAs were extracted using Trizol reagent (Takara, Dalian, China). First-strand cDNA was generated using a reverse transcription system kit (Invitrogen, Carlsbad, CA, USA). Real-time PCR was performed using the standard SYBR Green PCR kit protocol in the StepOne Plus system (Applied Biosystems, Foster City, CA, USA). For each sample, gene expression was normalized to 18S rRNA. The primer sequences used were as follows: GHET1, 5′-CCCCACAAATGAAGACACT-3′ (forward) and 5′-TTCCCAACACCCTATAAGAT-3′ (reverse); GAPDH, 5′-GGTCTCCTCTGACTTCAACA-3′ (forward) and 5′-GTGAGGGTCTCTCTCTTCCT-3′ (reverse); c-Myc, 5′-GGGCTTTATCTAACTCGCTGTA-3′ (forward) and 5′-GCTATGGGCAAAGTTTCGTG-3′ (reverse); 18S rRNA, 5′-ACACGGACAGGATTGACAGA-3′ (forward) and 5′-GGACATCTAAGGGCATCACA-3′ (reverse). The real-time PCR reactions were performed in triplicate. Relative RNA expression was calculated using the comparative Ct method .
5′ and 3′ RACE
We used the 5′ and 3′ RACE analysis to determine the transcriptional initiation and termination sites of GHET1 using a SMARTer™ RACE cDNA amplification kit (Clontech, Palo Alto, CA) according to the manufacturer's instructions. The gene-specific primers used for the PCR in the RACE analysis were 5′RACE, 5′-GCTTGCTGGGATTTACCGACGCTGGA-3′; 3′RACE, 5′-AACAGTAAGCAGAGTAAACAGACAACCCAC-3′.
cDNA encoding GHET1 was PCR-amplified using Pfu Ultra II Fusion HS DNA polymerase (Stratagene/Agilent Technologies, Palo Alto, CA, USA), and sub-cloned into the pcDNA3.1 vector (Invitrogen). The primers used were 5′-CCCAAGCTTACCAGAGAGCCGCCCAATC-3′ (forward) and 5′-CGGGATCCGCCATTCTTGCAGGAGTAAT-3′ (reverse).
siRNA synthesis and transfection
siRNAs specifically targeting GHET1 were synthesized by Invitrogen, siRNAs specifically targeting c-Myc were synthesized by GenePharma (Shanghai, China), and siRNAs specifically targeting IGF2B1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The siRNA sequences were 5′-GGUGAUCCAGACUCUGACCUU-3′ for c-Myc and 5′-CGGCAGGCATTAGAGATGAACAGCA-3′ for GHET1. Transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The transfected cells were harvested 48 or 72 h after transfection.
Cell proliferation assay
A total of approximately 3.0 × 103 gastric carcinoma cells were plated in 96-well plates. After 24 h culture, cell proliferation was assessed using Cell Counting Kit-8 (CCK-8; R&S Bio-Technology, Shanghai, China) according to the manufacturer's instructions. EdU immunofluorescence staining was performed using an EdU kit (Roche, Mannheim, Germany). All experiments were performed in triplicate. The cell proliferation curves were plotted using the absorbance at each time point.
Colony formation assay
Colony formation assays were performed as described previously . The experiments were performed in triplicate.
Total cell lysates were prepared in 1 × SDS buffer. Identical quantities of proteins were separated by SDS–PAGE and transferred onto nitrocellulose membranes. After incubation with antibodies specific for c-Myc, GAPDH (Santa Cruz Biotechnology) or IGF2BP1 (Cell Signaling Technology, Boston, MA, USA), the blots were incubated with goat anti-rabbit or anti-mouse secondary antibody (Santa Cruz Biotechnology) and visualized by enhanced chemiluminescence.
RNA immunoprecipitation (RIP)
RIP experiments were performed using the Magna RIP™ RNA-binding protein immunoprecipitation kit (Millipore, Bedford, MA, USA) according to the manufacturer's instructions. The antibody for RIP assays of IGF2BP1 (Cell Signaling Technology) was diluted 1:50. Co-precipitated RNAs were detected by quantitative RT-PCR.
RNA pull-down assay
GHET1 and its antisense RNA were in vitro transcribed and biotin-labeled using a biotin RNA labeling mix (Roche, Indianapolis, IN) and T7/SP6 RNA polymerase (Roche), treated with RNase-free DNase I (Roche) and purified using an RNeasy mini kit (Qiagen, Valencia, CA, USA). One milligram of protein from MKN45 cell extracts was mixed with 50 pmol biotinylated RNA, incubated with streptavidin agarose beads (Invitrogen), and washed three times with NaCl/Pi at room temperature. The retrieved proteins were detected using a standard western blotting technique.
Generation of cells stably transfected with GHET1
To obtain cell lines stably expressing GHET1, MKN45 cells were transfected with the pcDNA3.1-GHET1 plasmid and selected by treatment with neomycin (800 μg·mL−1) for 4 weeks. Stably over-expressing cell lines were identified by real-time PCR. Stable cell lines expressing the empty pcDNA3.1(+)plasmid were used as controls.
In vivo tumorigenesis assay
Cells from the GHET1 and the control vector stable expression cell lines were subcutaneously implanted into the flanks of nude mice (Shanghai Laboratory Animal Center of the Chinese Academy of Sciences, Shanghai, China). The experiments were approved by the Institutional Animal Care and Use Committee of the Second Military Medical University, Shanghai, China. Five million GHET1-over-expressing MKN45 cells (firefly luciferase-labeled) or control cells were injected into opposite armpits of nude mice. We analyzed primary tumor growth by measuring tumor length (L) and width (W), and calculated tumor volume according to the equation V =0.4 × LW2. Tumor growth was also monitored using the IVIS@ Lumina II system (Caliper Life Sciences, Hopkinton, MA, USA) 10 min after intraperitoneal injection of 4.0 mg luciferin in 50 μL saline.
For comparisons, Student's t-test (two-tailed), Wilcoxon signed-rank test, Pearson chi-square test, Pearson correlation analysis and Log-rank test were performed as indicated. All P values were two-sided and were obtained using the spss 18.0 software package (SPSS, Chicago, IL, USA). Differences were defined as statistically significant for P value < 0.05.
This work was supported by grants from the Science and Technology Commission of Shanghai Municipality, China (124119a1800).