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LEIGClong non-coding RNA acts as a tumor suppressor in gastric carcinoma by inhibiting the epithelial-to-mesenchymal transition
- Yuehua Han†1,
- Jun Ye†1,
- Dang Wu†2,
- Pin Wu2,
- Zhigang Chen2,
- Jian Chen3,
- Shunliang Gao3 and
- Jian Huang2, 4Email author
© Han et al.; licensee BioMed Central. 2014
Received: 11 June 2014
Accepted: 25 November 2014
Published: 11 December 2014
Long non-coding RNAs have been shown to have critical regulatory roles in cancer biology. However, the contributions of lncRNAs to gastric cancer remain largely unknown.
The differential expression of lncRNAs in gastric cancer and paired non-cancerous tissues were identified by microarray and validated using quantitative real-time PCR. Gastric samples from patients with gastric cancer were further analyzed for levels of a specifically downregulated lncRNA (termed as LEIGC).
We found that there were significantly lower levels of LEIGC expression in cancer tissue than in adjacent non-cancerous tissues in human gastric cancers (P < 0.01). Overexpression of LEIGC suppressed tumor growth and cell proliferation, and enhanced the sensitivity of gastric cancer cells to 5-fluorouracil (5-FU), whereas knockdown of LEIGC showed the opposite effect. We further demonstrated LEIGC functions by inhibiting the epithelial-to-mesenchymal transition (EMT) in gastric cancer.
Our data suggested that LEIGC is a tumor-suppressing lncRNA in gastric cancer, and led us to propose that lncRNAs may play important regulatory roles in cancer development and progression.
Gastric cancer is the fourth leading cause of cancer death, with a high mortality worldwide, especially in Asia [1, 2]. Unfortunately, gastric cancer is difficult to cure unless it is identified at an early stage, before it has begun to spread. The 5-year survival rate of gastric cancer patients remains poor, at approximately 40%, despite recent advances in surgical techniques and medical treatment [3, 4]. Metastasis is the main cause of death from such tumors. Thus, there is an urgent need to identify new molecular markers for early diagnosis, prediction of metastatic progression and prognosis of gastric cancer patients.
The human transcriptome comprises not only large numbers of protein-coding messenger RNAs (mRNAs), but also many non-protein coding transcripts that function as important regulatory molecules in tumor suppressor or oncogenic pathways . Non-coding RNAs are divided into short non-coding RNAs and long non-coding RNAs depending on their length. Long non-coding RNAs (lncRNAs) are defined as non-coding RNAs of more than 200 nucleotides in length, and are characterized by the complexity and diversity of their sequences and mechanisms of action . Recent deep transcriptome sequencing and microarray studies have revealed that 70–90% of the human genome is estimated to be transcribed into mostly non-protein-coding RNA . Increasing evidence indicates that lncRNAs exert important roles in a wide range of biological processes, including cell differentiation, chromatin remodeling, immune responses and tumorigenesis [6–8]. LncRNA levels are strongly associated with aberrant gene expression that may drive cancer development and progression , such as HOTAIR in non-small cell lung cancer (NSCLC) , PRNCR1 (also known as PCAT8) and PCGEM1 in prostate cancer , and MEG3 in cervical cancer and meningiomas [12, 13]. Thus, differential expression of lncRNAs may be profiled to aid cancer diagnosis, prognosis and selection of potential therapeutics.
Although lncRNAs play important roles in human diseases, the mechanism through which they contribute to cancer development is still largely unknown. LncRNAs can regulate critical cancer pathways at a transcriptional, post-transcriptional and epigenetic level . Mounting evidence suggests that a major role of lncRNAs is to act as modular scaffolds for protein-chromatin interactions . Several lncRNAs can control gene expression by direct recruitment of histone-modifying enzymes to chromatin [6, 15]. Chromatin modification and DNA methylation are crucial epigenetic events that are fundamentally disturbed during the development of cancer. LncRNAs can also affect protein-coding transcript response to different biological processes .
However, there are only preliminary studies on the role of lncRNAs in gastric cancer [17–19], and the overall pathophysiological contributions of lncRNAs to gastric cancer remain largely unknown. A current estimate of the lncRNA gene number in the human genome ranges from 8000–20,000 unique lncRNAs [20, 21], suggesting lncRNAs constitute a large yet undiscovered part of normal cellular networks that may be disrupted in cancer. Therefore, it is of great importance to explore the molecular mechanisms of lncRNAs in gastric cancer development and progression. In this study, we aimed to investigate the expression pattern and clinicopathological implications of lncRNAs in gastric cancer tissues. We identified a new specific differentially-expressed lncRNA (termed LEIGC), which was downregulated in gastric cancer tissues compared with adjacent non-cancerous tissues. Then we performed gain- and loss-of-function studies to determine the effect of LEIGC on tumor growth, cell proliferation, and migration, and showed that LEIGC suppressed tumor growth, cell proliferation and EMT in gastric cancer, and increased the sensitivity of gastric cancer cells to 5-FU.
Human gastric cancer cell lines, MGC-803, AGS, SGC-7901 were purchased from the cell bank of China Academy of Medical Science (China). Cells were cultured in RPMI 1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and maintained at 37°C with 5% CO2.
LncRNA expression microarray analysis
Total RNA of gastric cancer tissues and paired normal tissues were extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and treated with RNase-free DNase I (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. The quantity and quality of RNA was evaluated using a Nanodrop spectrophotometer (Thermo Scientific, Worcester, MA, USA). The lncRNA expression profile of each sample was examined using a lncRNA expression microarray (SurePrint Human Gene Expression Microarray Kit, Agilent technologies, Santa Clara, CA, USA). The BROAD Institute database was used in the genesis of the array. After hybridization and washing, the processed slides were scanned with the Agilent Microarray Scanner (Agilent technologies Santa Clara, CA, USA). Raw data were extracted as pair files using Feature Extraction software 10.7 (Agilent technologies). A fold change of ≥ 2.0 or <0.5 (P ≤ 0.05) was set as a threshold for up- and downregulated genes, respectively, and data were presented as mean ± SD. Raw data were normalized by a Quantile algorithm using Gene Spring Software 11.0 (Agilent technologies). Hierarchical clustering was performed based on differentially-expressed lncRNAs using Cluster Treeview software from Stanford University.
Structural analysis of LEIGC
We used the BLAT program of the University of California Santa Cruz (UCSC), Genome Browser, BLAST, and MAP VIEW of the NCBI to analyze the gene sequence and chromosomal location of LEIGC.
Quantitative real time PCR analysis
Primer sequences used in qRT-PCR
F: 5’- agg ata cgt aag aaa cac ttc tgt -3’
R: 5’- tgt ctt ggt tta aca acc ga -3’
F: 5’- acc cca cat cct tct cac tg −3
R: 5’- tac aaa aac cca cgc aga ca −3
F:5’- aca cac aca cac cca cag ag −3
R:5’- aaa tga ttt ggc agc aat gt −3
F: 5’- gca caa cca agt gca gaa ga -3’
R: 5’- cat ttg cag att gag gct ga -3’
F: 5’- tgc tct tgc tgt ttc ttc gg-3’
R: 5’- tgc ccc att cgt tca agt ag-3’
F: 5’- tgg atg gac ctt atg ttg ct -3’
R: 5’- aac acc tgt ctt ggg atc aa -3’
F: 5’-gga gtc cgc agt ctt acg ag-3’
R: 5’-cca gct tga ggg tct gaa tc-3’
F: 5’-ctt agc acc cct ggc caa g-3’
R:5’-gat gtt ctg gag agc ccc g-3’
Cycling conditions were 10 min at 95°C for initial denaturation, followed by 40 cycles of 15 sec at 95°C for denaturation, 30 sec at 60°C for combined annealing and 30 sec at 72°C for primer extension. Each sample was run in triplicate and the gene expression levels were normalized to that of GAPDH expression.
Establishment of lncRNA or lncRNA-shRNA stable cells
LEIGC vector (LV6-Puro) or LEIGC-shRNA vector (pGLV2-U6-Puro) (Additional file 1: Figure S1) and scrambled shRNA or non-related lncRNA vector lentiviral particles (GenePharma Tech, Shanghai, China) were transfected into MGC-803 cells. Cells were selected with 5 μg/ml puromycin (Sangon Biotech, Shanghai, China) at 48 h after transfection. The overexpression and knockdown efficiencies were verified by qRT-PCR.
In vitromotility assay
Transwell insert chambers with 8-μm porous membranes (Corning Incorporated, NY, USA) were used for motility assays. Cells were washed three times with PBS and 5 × 104 cells in serum-free media were added to the top chamber. The bottom chamber was filled with RPMI 1640 medium containing 15% FBS. Cells were incubated for 24 h at 37°C in 5% CO2. To quantify migrating cells, cells in the top chamber were removed using a cotton-tipped swab, and the migrated cells were fixed in methanol and stained with 0.1% crystal violet.
Cell growth assay
Cells were seeded at a density of 3 × 103 cells/well in a 96-well plate containing 0.2 ml RPMI 1640 medium with 10% FBS. Then 20 μl MTS (3-[4,5-dimethylthiazol-2-yl] -5-[3-carboxmethoxy phenyl]-2-[4-sulfophenyl]-2H-tetrazolium salt) (Promega) reagent was added to each well and the cells were incubated at 37°C for 4 h. The OD values were measured at 490 nm on a microplate reader (Bio-Rad, Hercules, CA, USA) and assessed daily for 7 days.
Colony formation assay
The proliferative ability of cells was tested in colony formation assays. Approximately 300 cells were seeded into each well of a 12-well plate. After incubation at 37°C for 14 days, the cells were washed twice with PBS, fixed with methanol and then stained with 0.1% crystal violet. The number of colonies containing more than 300 cells was counted under a microscope.
A total of 1 × 106 cells suspended in 100 μl RPMI 1640 were implanted into the hindquarters of 4-week-old female NOD/SCID mice to assess their ability to initiate tumor xenografts. The use and care of experimental animal was approved by Institutional Animal Care and Use Committee of Zhejiang Chinese Medical University. Tumors were measured weekly and their volume calculated as length × width × width/2 .
Total protein from cells was lysed using M-PER Mammalian Protein Extraction Reagent (Thermo) supplemented with a protease inhibitor cocktail (Sigma, St Louis, MO, USA). Samples were denatured, and equal amounts of protein were subjected to SDS-PAGE, and then transferred to nitrocellulose membrane. After blocking with 5% non-fat milk in TBST for 60 min, membranes were incubated with primary antibody dissolved in 5% bovine serum albumin in TBST overnight at 4°C. The following primary antibodies were used: anti-human- E-cadherin (1:2000, 24E10; Cell Signaling Technology, Danvers, MA, USA), anti-human-Vimentin (1:2000, D21H3; Cell Signaling Technology), anti-human-Snail (1:1000, C15D3; Cell Signaling Technology), anti-human-Twist (1:1000; Cell Signaling Technology), anti-human-Zeb (1:1000, D80D3; Cell Signaling Technology), and anti-human-Slug (1:1000, C19G7; Cell Signaling). Human GAPDH (1:5000; KangChen, Shanghai, China) was used as an internal reference.
Cell viability assay
MGC-803 cells were transfected with LEIGC vector or LEIGC shRNA vector and scrambled shRNA or non-related lncRNA vector, and incubated for 24 h. Then cells were reseeded in 96-well plates and treated with 5-FU (NanTong Pharmaceutical Factory, China) at different concentrations for 48 h. The cell viability was determined by the MTT assay: 20 μl of MTT (Sigma) per well was added and incubated at 37°C for 4 h. The reaction was stopped by adding 200 μl of dimethyl sulfoxide (DMSO) to each well followed by measuring the absorbance at 570 nm on a microplate reader (Bio-Rad, USA) for the indicated time periods at 37°C to determine the individual IC50 values (50% cell growth inhibitory concentrations).
Clinical gastric cancer sample analysis
Clinicopathological features of gastric cancer patients
Cases (n = 35)
Lymph node metastasis
M0 (NM group)
M1 (M group)
Data are presented as the means ± standard error of the mean (SEM). All statistical analyses were performed using SPSS 16.0 software. The qRT-PCR results from paired clinical samples were analyzed by a two-tail paired Student’s t-test and the other results by a two-tail unpaired Student’s t-test. P values of <0.05 indicated statistical significance.
Expression of LEIGCin gastric cancer tissues
Structural analysis of LEIGC
LEIGC suppresses tumor growth in vitro
LEIGC inhibits migration of gastric cancer cells in vitro
LEIGC knockdown promotes tumor progression in vivo
LEIGCenhances chemosensitivity to 5-FU in gastric cancer
LEIGCis a novel factor that prevents EMT in gastric cancer
To confirm that LEIGC knockdown in MGC-803 cells resulted in a mesenchymal phenotype, we analyzed the gene expression profiles of LEIGC-overexpressing cells versus knockdown MGC-803 cells. As shown in Figure 7, the epithelial cell-related gene CDH1 was significantly downregulated, whereas several mesenchymal cell markers (such as snail, slug, zeb, and twist) were significantly upregulated in LEIGC knockdown cells compared with LEIGC-overexpressing cells (Figure 7F). We further examined EMT-associated protein expression in MGC-803 cells by western blotting. As shown in Figure 7G, LEIGC knockdown cells demonstrated lower expression of E-cadherin and higher expression of Vimentin, Snail, Slug, Zeb, and Twist. Overexpression of LEIGC showed the opposite effect. These data indicated that LEIGC was a potent EMT inhibitor in gastric cancer cells.
Over the past few years, hundreds of lncRNAs have been shown to play important roles in both transcription and post-transcriptional processes. Studies have reported that lncRNA dysfunctions are associated with a broad range of human tumors, including those of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), HOX antisense intergenic RNA (HOTAIR), antisense non-coding RNA in the INK4 locus (ANRIL), and lncRNA-p21 [10, 24]. lncRNAs are aberrantly expressed in many types of cancers [25, 26]. However, the potential roles of lncRNAs in human cancers are not well understood. In this study, we verified that LEIGC was significantly downregulated in gastric cancer tissues compared with paired non-cancerous tissues. Furthermore, our results indicated that LEIGC inhibited tumor growth, proliferation, migration, and EMT in gastric cancer cells. Hence, our results also suggest that LEIGC is a putative tumor/metastasis suppressor in gastric cancer.
Recently, many studies have shown that lncRNAs have important roles in the regulation of numerous biological processes in cancer, including tumor proliferation, migration, angiogenesis, and EMT. Altered expression of lncRNAs has been documented in different human cancer types, prompting increased interest in their use as biomarkers for diagnosis and prognosis as well as potential therapeutic targets . For example, a study demonstrated that HULC was significantly overexpressed in gastric cancer cell lines and gastric cancer tissues compared with normal tissues, and its overexpression was correlated with distant metastasis and lymph node metastasis . Knockdown of HULC inhibited proliferation, invasion and EMT, and promoted cell apoptosis in SGC-7901 gastric cancer cells. Recently, increased levels of HOTAIR in primary breast tumors were shown to correlate with breast cancer invasiveness and metastasis . HOTAIR bridges together the PRC2 complex with the LSD1 H3K4 demethylase complex, and recruits both complexes to target genes to coordinately alter several histone modifications and enforce gene silencing. The increased expression of HOTAIR in human gastric cancers was associated with venous invasion, lymph node metastases and a lower overall survival rate [9, 15].
To explore the exact mechanisms of LEIGC in gastric cancer, we used gene transfection experiments to overexpress and silence LEIGC in MGC-803 gastric cancer cells. The key event for malignant tumor progression is metastasis, which is based on tumor cell migration and invasion. Metastasis accounts for the majority of gastric cancer-related mortality, but the mechanism of the metastatic process in gastric cancer is very complex, and still not completely understood. EMT was originally recognized as a critical step to metazoan embryogenesis and in defining structures during organ development . During the last decade, a number of studies have associated EMT with cancer progression and metastasis in gastric cancer. In our transwell assay, knockdown of LEIGC dramatically promoted cell migration in gastric cancer cells (Figure 4). We found LEIGC silencing was associated with features typical of EMT, including the conversion of the cobblestone-like epithelial morphology to spindle-shape mesenchymal morphology, reduced expression of CDH1, and increased expression of snail, slug, twist and zeb. Consistent with the observed morphological changes, some hallmark proteins of epithelial cells were lost or reduced during the transition, such as E-cadherin. In contrast, the mesenchymal protein vimentin was upregulated. It is well known that E-cadherin plays a critical role in the suppression of tumor invasion. Most epithelial cancers display downregulated or inactivated E-cadherin [28, 29]. It has been shown that the restoration of functional E-cadherin suppresses invasion in many tumor types. Snail, Twist, Slug and Zeb associated with EMT have all been shown to target E-boxes on the E-cadherin promoter, repressing its expression [30, 31]. We observed that snail, slug, twist and zeb genes and corresponding proteins were highly elevated in LEIGC knockdown cells (Figure 7). However, overexpression of LEIGC resulted in the opposite effect in MGC-803 cells. Taken together, these data indicated that LEIGC is a critical regulator in preventing EMT in gastric cancer. However, in our study, no significant correlation was found between LEIGC expression and distant tumor metastasis or lymph node metastasis in gastric cancer (data not shown). This might be because of the low number of gastric patients in our study.
In summary, our data provides evidence that may mechanistically link the expression of LEIGC to the proliferation and migration of gastric cancer cells. We demonstrate that LEIGC functions as a tumor suppressor lncRNA in gastric cancer by inhibiting EMT, and propose that lncRNAs may play important regulatory roles in cancer development and progression. Further analysis and investigation of the mechanisms of LEIGC in the molecular etiology of gastric cancer will provide lncRNA-directed diagnostic and therapeutic tools against this deadly disease.
This work was supported by grants from Zhejiang Provincial Natural Science Foundation of China (No. LQ12H16009), the Science and Technology Bureau of Zhejiang Province (No. 2013C33137), and Science and Technology Project of the health department of Zhejiang Province (No. 2008A092). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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