Background
Gastric cancer (GC) is the third leading cause of cancer-related death and the fifth most frequently diagnosed cancer worldwide, with 1,000,000 new gastric cancer cases and 783,000 death predicted in 2018 [
1,
2]. Notably, the incidence rates are dramatically increased in Eastern Asia, which has the highest incidence level worldwide in both sexes [
2,
3]. The majority of newly diagnosed GC patients have reached an advanced stage due to absence of sensitive and specific biomarkers [
4,
5]. Consequently, there is an urgent need to further GC pathogenesis and identify useful markers implicated in GC.
Long non-coding RNAs (lncRNAs) are newly-discovered transcripts larger than 200 nucleotides [
6‐
8]. Recent research has reported that lncRNAs participate in numerous biological activities, such as apoptosis, cell differentiation and metastasis, thus contributing to cancer progression [
9‐
11]. Also, the interplay between lncRNAs and cellular molecules, such as protein, RNA and DNA, can lead to ectopic expression of critical genes [
12‐
15]. Therefore, studies of potential molecules associated with gastric tumourigenesis represent an attempt to identify the possible application of lncRNAs as effective markers in GC.
The novel lncRNA MNX1-AS1 originally identified as a highly-expressed gene in colorectal cancer (CRC) is also known as CCAT5 [
16,
17]. Wang et al. demonstrated that CCAT5 can facilitate CRC progression by interacting with STAT3 [
18]. Another research by Ye et al. reported that overexpression of MNX1-AS1 is activated by E2F1 in CRC cells and that MNX1-AS1 can increase SEC61A1 expression by sponging miR-218-5p to facilitate CRC development [
19]. In addition, CRC patients in high-MNX1-AS1 expression group exhibited remarkably worse prognostic outcome than those in low-MNX1-AS1 expression group, displaying the robust predictive value of MNX1-AS1 in CRC [
19]. Mounting evidence has also shown the presence of MNX1-AS1 overexpression in multiple cancers, including glioblastoma, cervical cancer, GC, ovarian cancer and breast cancer [
20‐
23]. It is reported that MNX1-AS1 exerts promoting effects on the proliferative capacities of cancer cells in both glioblastoma and ovarian cancer [
20,
21] In breast cancer, MNX1-AS1 has been characterized as an oncogene to induce epithelial-mesenchymal transition (EMT) and activate the AKT/mTOR signal pathways [
22]. Moreover, MNX1-AS1 facilitated cervical cancer progression through activating mitogen-activated protein kinase (MAPK) pathway [
23]. An earlier research reported by Zhang and his colleagues revealed that increased MNX1-AS1 level predicts poor prognosis in patients with GC [
23]. Recently, Ma et al. demonstrated that MNX1-AS1 can exert promotive effects on GC migration and invasion by repressing CDKN1A [
24]. However, diverse mechanisms behind MNX1-AS1 dysregulation remain largely unknown in GC. The present study is designed to comprehensively investigate MNX1-AS1-mediated mechanistic models in GC progression.
For the present research, RNA sequencing data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets were analysed to find deregulated expression of lncRNAs, which were correlated with GC progression. Among the significantly overexpressed lncRNAs, MNX1-AS1 was selected for further investigation, and the results demonstrated that MNX1-AS1 was significantly amplified in GC and its upregulation frequently predicted poor clinical outcomes. Also, TEA domain family member 4 (TEAD4) activated MNX1-AS1 can mediate the expression level of BTG anti-proliferation factor 2 (BTG2) and BCL2 apoptosis regulator (BCL2) in GC cells. Our findings may illuminate the effectors involved in MNX1-AS1 overexpression and further clarify the underlying mechanism by which MNX1-AS1 mediates the malignant phenotype of GC cells.
Methods
LncRNA expression profile analysis
Gene expression data of GC were obtained from TCGA and GEO (GSE62254) datasets. The BAM files and normalized probe-level intensity files were downloaded from TCGA and GEO databases. The probe sequences were downloaded from GEO or microarray manufacturers.
Collection of GC tissues
We collected tissue specimens from 174 GC patients at the People’s Hospital of Jiangsu Province (Nanjing, Jiangsu, China). All the patients signed an informed consents and received no chemotherapy before the operation. 2010 tumour–node–metastasis (TNM) staging recommended by American Joint Committee on Cancer system (AJCC 7th edition) was used to classify tumour stages. This study was approved by the Human Ethics Committee of Nanjing Medical University (Nanjing, Jiangsu, China) and implemented in accordance with the Helsinki Declaration of Principles.
Cell lines culture
We purchased the MGC803, SGC7901, BGC823, HEK-293 T and GES-1 cell lines from Shanghai Cell Bank Library of the Chinese Academy of Sciences (Shanghai, China). SGC7901, SGC803, GES-1 and HEK-293 T cells were cultured in RPMI DMEM (Invitrogen, Shanghai, China) with 10% foetal bovine serum (FBS) at 37 °C in 5% CO2. BGC823 and GES-1 cells were cultured in RPMI1640 medium supplemented with 10% FBS at 37 °C in 5% CO2.
RNA extraction and qRT-PCR assays
Total RNA was isolated with TRIZOL reagent (Invitrogen, Grand Island, NY, USA) according to the product descritption. One microliter total of RNA was reverse-transcribed into cDNA using Primescript RT reagent kit (Takara, Dalian, China). cDNA was quantified by RT-PCR and the data were acquired with SYBR Green (Takara, Dalian, China) using Applied Biosystems 7500 instrument. GAPDH was used as an internal control. The primers are listed in Additional file 4: Table S3.
Cell transfection
The plasmid vectors were purified with a DNA Midiprep kit (Qiagen, Hilden, Germany). Three siRNAs and a scrambled RNA construct were designed to prevent the off-target effects and ensure the efficiency of interference. MiRNA mimics and their corresponding miRNA inhibitors were purchased from Ribobio (Guangzhou, China). Cells were transfected using Lipofectamine 3000 (Invitrogen). The sequences of siRNA and shRNA are presented in Additional file
4: Table S3.
Cell migration and invasion assays
The details of cell migration and invasion assays are described in Additional file
5.
Cell proliferation assay
Cell proliferation ability was examined using a Proliferation Reagent Kit I (MTT) (Roche, Basel, Switzerland). Details are available in Supplementary Materials and Methods.
Flow cytometry assay
We analysed cell cycle and apoptosis using flow cytometry assays based on the manufacturer’s protocol. Details of Flow cytometry assay are supplied in Additional file
5.
Western blotting
Protein extraction and quantities were poformed according to the description in Additional file
5. All antibodies are listed in Additional file
4: Table S3.
The programme was approved by the Animal Experimental Ethics Committee of Nanjing Medical University. All experimental procedures involving animals were performed in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals. Details of Tumour formation assay are described in Additional file
5.
Immunohistochemical (IHC) analysis
For hematoxylin-eosin stain (HE) staining, tumour tissues from mice were embedded and sectioned, and then stained with haematoxylin and eosin. For immunohistochemical studies, we incubated the samples with antibodies against Ki67. After washing the sample with PBS, the second antibody was added. The signal was amplified and visualized with 3′-diaminobenzidine chromogen (DAB), and then counterstained with haematoxylin.
RNA immunoprecipitation(RIP)
An EZMagna RNA Immunoprecipitation (RIP) Kit (Millipore) was used according to the manufacturer’s protocol. Details of the RIP experiment are obtained in Additional file
5. The detailed information of antibodies are listed in Additional file
4: Table S3.
Chromatin immunoprecipitation assays(ChIP)
ChIP assays were performed with a MagnaChIP kit (Millipore). The specific method of ChIp is shown in Additional file
5. The primers are listed in Additional file
4: Table S3.
Luciferase assays
The TEAD4 binding sequence on the promoter region of MNX1-AS1 was determined by JASPAR (
http://jaspar.genereg.net/). The details of luciferase assays are described in Additional file
5. Each experiment was repeated three times.
Fluorescence in situ hybridization (FISH) and subcellular separation
The FISH and subcellular separation assay of SGC-7901 and MGC-803 cells were conducted according to the method described in Additional file
5. The probe sequences are listed in the Additional file
4: Table S3.
Statistical analysis
The significance of the differences between the groups was evaluated using Student’s t test, Wilcoxon test or χ2 test. The data were analysed with SPSS 17.0 software (IBM), and the double-sided p value was calculated, p values less than 0.05 were considered to indicate a significant difference.
Discussion
A growing body of research has illuminated that dysregulated lncRNAs may mediate cancer progression [
41‐
43]. Recent evidence of the roles of lncRNAs in cancer pathogenesis has added to our knowledge of cancer biology. Although dysregulation of certain lncRNAs in gastric tumourigenesis is a recognized phenomenon, the functional mechanisms of most lncRNAs still remain undetermined in human GC.
Currently, the expression pattern and functional role of lncRNA MNX1-AS1 in diverse tumours have been gradually identified [
22,
24,
44,
45] Yang et al. found that overexpression of MNX1-AS1 exerted tumour-promoting roles in lung adenocarcinoma [
45]. A current study by Ji et al. showed that enrichment of MNX1-AS1 is strongly linked to TNM stage in hepatocellular carcinoma (HCC) patients [
46]. More interestingly, MNX1-AS1 can sponge miR-218-5p to activate the expression of COMMD8, thus facilitating malignant properties of HCC [
46]. Previous data provided by Ma et al. showed that impaired MNX1-AS1 expression can regulate CDKN1A expression to suppress invasive capacities of GC cells [
24]. To the best of our knowledge, few studies have reported the use of in vitro (let alone in vivo) assays to explore the regulatory correlation between lncRNA MNX1-AS1 and GC, or tried to clarify the upstream regulation of lncRNAs. More studies in epigenetics may shed new light on cancer pathogenesis.
In this study, MNX1-AS1 expression was firstly detected via microarray analysis, and further determined in 174 paired GC tissue samples. Overexpressed MNX1-AS1 promoted GC cell growth and invasion, suggesting its critical value in GC patients. Current evidence has shown that TFs contribute to lncRNA dysregulation in various cancers [
25,
47]. TEAD4, a critical member of the TEA domain (TEAD) family, has been identified as an oncogenic regulator in many cancers, including GC [
48,
49]. Our data showed that MNX1-AS1 overexpression could be activated by TEAD4.
To clarify the regulatory mechanism of lncRNA MNX1-AS1 in GC, we used RNA-sequencing experiments to investigate potential target genes. It was reported in previous studies that epigenetic alterations such as aberrant histone modifications can participate in cancer development and progression [
50‐
52]. Moreover, lncRNAs have been shown to cooperate with chromatin-modifying enzymes, thus activating epigenetic activation or gene silencing [
52]. EZH2, an important member of PRC2, could mediate H3K27me3 and exert oncogenic functions through repressing tumour suppressors [
53,
54]. For this study, we found that MNX1-AS1 mainly localized in the nucleus, indicating that MNX1-AS1 may exert its oncogenic effects at transcriptional level. Mechanistic investigations elucidated that lncRNA MNX1-AS1 can recruit EZH2 and H3K27me3 to the promoter of BTG2 in the nucleus, thus partially silencing BTG2 expression and mediating oncogenic properties in GC.
Additionally, we also attach great importance to lncRNA-mediated post-transcriptional processing. For instance, our previous research has reported that linc00346 can function as a ceRNA to sponge miR-34a-5p in GC cells [
27]. In the present research, both bioinformatic analyses and luciferase reporter assays demonstrated that lncRNA MNX1-AS1 functions as a ceRNA for miR-6785-5p, thereby contributing to the depression of its endogenous target gene. In addition, miR-6785-5p upregulation impaired GC cell proliferation and induce cell apoptosis, while miR-6785-5p knockdown elicited the opposite effects. Interestingly, both RNA-seq data and subsequent verification assays revealed that BCL2 was obviously downregulated in MNX1-AS1-depleted-GC cells. Prediction results from miRanda also revealed that BCL2 was likely to bind with miR-6785-5p. Thus, we proposed the hypothesis that BCL2 may be a candidate target of miR-6785-5p, To confirm this hypothesis, we used luciferase reporter assays and showed that miR-6785-5p targeted BCL2 mRNA at its 3’UTR.
Collectively, we have uncovered that MNX1-AS1 is a novel lncRNA correlated with GC tumourigenesis and progression. TEAD4, a critical oncogenic transcription factor, may be involved in promoting the transcription of lncRNA MNX1-AS1 via binding to the promoter region of MNX1-AS1. In addition, we identified a molecular mechanism underlying GC development that involves lncRNA MNX1-AS1/EZH2/BTG2 and MNX1-AS1/miR-6785-5p/BCL2 axes (Fig.
10).
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