Background
Gastric cancer (GC) is the third leading cause of cancer-related death worldwide due to the combination of its high incidence and a lack of effective treatment options [
1]. GC is often diagnosed in the middle- or late-stage and is accompanied by malignant proliferation and metastasis in most patients. Even with significant advances in surgical techniques, diagnosis and molecular targeting therapy, the prognosis of advanced-stage patients remains very poor [
2‐
4]. As such, a better understanding of the molecular mechanism of GC progression is necessary to provide potential biomarkers and targets for improving the diagnosis and treatment of GC.
To date, most studies have mainly focused on protein-coding genes. However, human genome sequencing data reveals that protein-coding sequences occupy less than 2% of the human genome, and 98% are non-coding RNAs [
5,
6]. Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs with transcripts that are > 200 nt long and have limited or no protein-coding potential [
7]. Despite not encoding proteins, lncRNAs have been revealed to play essential roles in tumorigenesis [
7] and regulating the expression of potential target genes at the epigenetic, transcriptional, and post-transcriptional levels [
8,
9]. LncRNAs also play key roles in critical biological processes, such as chromosome imprinting, stem cell differentiation, immune response, tumorigenesis, and chemotherapy resistance [
10‐
12]. Recently, numerous lncRNAs were revealed to be associated with human diseases, especially cancer [
13]. However, the role of lncRNAs in the development of GC is explicitly not well understood.
In this study, we first identified a novel GC-associated lncRNA, LINC01050, which is activated by c-Myc. We found that LINC01050 was significantly up-regulated in GC tissues compared with the corresponding non-tumor tissues, and its expression may serve as a potential independent predictor for overall survival in GC. Moreover, we determined that LINC01050 regulated GC progression and metastasis by functioning as a competing endogenous RNA (ceRNA) for miR-7161-3p, thereby preventing the latter’s association with its target SPZ1. Our data indicate that LINC01050 plays a critical role in GC progression and is a potential candidate for GC diagnosis and treatment.
Materials and methods
Data treating
The data of RNA expression profiles for stomach adenocarcinoma (STAD) were downloaded from Xena platform [
14], including 375 STAD tissues and 32 non-tumor tissues. The lncRNAs were annotated by the human gene annotation files (GRCh38.90), which was downloaded from the Ensembl database (
https://asia.ensembl.org). The lncRNAs were defined as 3-prime overlapping ncRNAs, anti-sense RNAs, bidirectional promoter lncRNAs, long intergenic non-coding RNAs (lincRNAs), macro lncRNAs, non-coding, processed transcript, sense intronic, and sense overlapping. The lncRNA expression data were analyzed by the R/Bioconductor package DESeq2 [
15]. The clinical data information for STAD were collected using R/Bioconductor package TCGAbiolinks [
16].
Human tissue samples
Tissue from 29 GC cases was obtained with the written consent of patients who underwent surgery at the First Affiliated Hospital of Wenzhou Medical University. The Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University approved this study.
Cell culture
The human GC cell lines (AGS, BGC-823, and KATO III) and the HEK293T cell line were purchased from the Typical Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The AGS, BGC823, and KATO III cells were cultured in RPMI 1640 (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, St Louis, MO, USA). The HEK293T cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies) supplemented with 10% FBS. The cells were cultured in a humidified 37 °C incubator supplemented with 5% CO2.
Cell transfection
Transfection was performed using Lipofectamine 3000 (Life Technologies) according to the manufacturer’s protocol. The miR-7161-3p mimic, LINC01050 siRNA, c-Myc siRNA, SPZ1 siRNA, and scramble siRNA (si-NC) were purchased from Guangzhou Ruibo Biotechnology Co., Ltd. (Guangzhou, China). The nucleotide sequences of the siRNAs are listed in Additional file
1: Table S1.
Lentiviral vector construction and transduction
The human LINC01050 transcript cDNA was amplified in the BGC-823 cells and was cloned into the lentiviral vector pLVX-puro by digesting it with EcoRI and BamHI. A short hairpin RNA directed against LINC01050 (sh-LINC01050) was inserted into the pLKO.1 puro vector that was digested with AgeI and EcoRI. The lentiviruses were generated by the transient transfection of the transfer vector and three packaging vectors (pMDLg/pRRE, pRSV-REV, and pCMV-VSVG) into HEK293T cells. The GC cells were transduced with lentiviruses expressing LINC01050, sh-LINC01050, or the negative control.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
The total RNA was extracted from the cells using the TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA), and 1 μg of total RNA was used for cDNA synthesis using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. The expression levels of LINC01050, miR-7161-3p, SPZ1, and c-Myc were evaluated by qRT-PCR using SYBR Premix ExTaq (Takara, Japan) and the QuantStudio 5 real-time PCR system (Applied Biosystems, Warrington, UK). After an initial activation at 95 °C for 30 s, 40 PCR cycles were performed using the following conditions: denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 34 s. The U6 gene was used to normalize the expression level of miR-7161-3p. GAPDH was used to normalize the expression levels of LINC01050, c-Myc, and SPZ1. The specific PCR primers and RT primers are presented in Additional file
1: Table S2.
Isolation of cytoplasmic and nuclear RNA
Cytoplasmic and nuclear RNA was isolated using a PARIS Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. The expression level of LINC01050 in the cytoplasm and nucleus was detected by qRT-PCR.
Cell proliferation assays
Cell proliferation was assessed using the Cell Counting Kit-8 (CCK8) and ethynyl deoxyuridine (EdU) incorporation assays. After transfecting the cells with the siRNAs or miRNA mimics for 24 h, the GC cells were trypsinized and seeded into 96-well plates in a volume of 100 μl of complete medium (3000 cells/well). At 0, 24, 48, and 72 h after plating, 10 μl of the CCK8 solution (Dojindo, Japan) was added to each well. After an 4 h incubation, each well was measured at 450 nm according to the manufacturer’s instructions. EdU cell proliferation staining was performed using the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488 (Beyotime, China). Briefly, the cells were incubated with EdU for 2 h, fixed with 4% paraformaldehyde and permeated with 0.3% Triton X-100. Then the cells were incubated with the Click Reaction Mixture for 30 min at room temperature in the dark and stained with Hoechst. The stained cells were photographed by fluorescence microscopy (Leica, Wetzlar, Germany).
The GC cells transfected with si-LINC01050 were plated in 6-well plates at a density of 2000 cells/well for the plate colony formation assay. After 2 weeks, the colonies were fixed for 30 min with 4% paraformaldehyde and were stained for 15 min with 0.1% crystal violet. The plate colony formation was determined by counting the number of colonies. All the experiments were repeated three times.
Western blot analysis
The GC cells were lysed in RIPA buffer (Thermo Fisher Scientific) supplemented with protease and phosphatase inhibitors (Thermo Fisher Scientific). The total protein concentration was measured by the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. The total cellular protein was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Subsequently, the electrophoresed proteins were transferred to a polyvinylidene fluoride membrane and were blocked with 5% skimmed milk for 2 h at room temperature. Then, the membrane was incubated with the primary antibodies overnight at 4 °C and the secondary antibody for 1 h at room temperature. The specific bands were detected using an enhanced chemiluminescence detection system (Bio-Rad, California, CA, USA). The following primary antibodies were used: mouse anti-vimentin (diluted 1:5000; Cat. #550513; BD Biosciences, San Jose, CA, USA); rabbit anti-E-cadherin (diluted 1:1000; Cat. #3195S; Cell Signaling Technology, Danvers, MA, USA); rabbit anti-Cleaved PARP1 (diluted 1:1000; Cat. # ab32064; abcam); rabbit anti-Cleaved Caspase-3 (diluted 1:1000; Cat. #9664; Cell Signaling Technology); mouse anti-Bcl-2 (diluted 1:1000; Cat. #15071; Cell Signaling Technology); mouse anti-Bax (diluted 1:1000; Cat. #89477; Cell Signaling Technology); rabbit anti-GAPDH (diluted 1:1000; Cat. #5174S; Cell Signaling Technology); rabbit anti-SPZ1 (diluted 1:1000; Cat. #DF8886; Affinity Biosciences, OH, USA); rabbit anti-AGO2 (diluted 1:1000; Cat. # ab186733; abcam); and rabbit anti-c-Myc (diluted 1:1000; Cat. #18583S; Cell Signaling Technology).
Cell migration and invasion assays
The cell migration and invasion assays were performed using a Transwell chamber (Costar; Corning Incorporated, Cambridge, MA, USA) according to the manufacturer’s instructions. For the migration assay, the cells (1.5 × 105/200 μL) were seeded onto the Transwell filter membrane chambers in cell culture medium without FBS. Medium supplemented with 20% FBS was added to the lower chambers as a chemoattractant. After an 36 h incubation, the cells on the upper membrane were removed. The bottom surface was fixed with 4% paraformaldehyde for 20 min and was stained with a 0.1% crystal violet solution for 15 min. The number of cells that migrated to the lower chamber was counted (the fields were randomly selected under a light microscope at a magnification of × 10). For the invasion assay, the upper membranes were precoated with 10 μL of Matrigel (4.53 mg/mL; BD Biosciences, San Jose, CA, USA) before the process described above was carried out.
Apoptosis assays
The KATO III and BGC823 cells were transfected with si-LINC01050 or si-NC for 48 h. Cell apoptosis was measured using an Annexin V-FITC/ Propidium iodide (PI) apoptosis detection kit (Multi Sciences, Hangzhou, China) according to the manufacturer’s protocol. After double staining with Annexin V-FITC (5 μL) and PI (10 μL), the cells were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).
Luciferase assay
To generate the LINC01050 promoter construct, the fragment (between − 740 and − 2000 bp) was amplified from HEK293T DNA and inserted into the pGL3-basic luciferase reporter vector (Promega, Madison, WI, USA). To construct the LINC01050 promoter mutation vector, the c-Myc binding site sequence was deleted in the corresponding LINC01050 promoter construct using the QuikChange Lightning Site-Directed Mutagenesis Kit (STRATAGENE, USA). The deletion was confirmed by sequencing. The pIRES2-c-Myc and pIRES2-vector were individually co-transfected into HEK293T cells together with the pGL3-based construct containing the LINC01050 WT or c-Myc deletion promoter sequences plus the Renilla plasmid (RL-SV40).
The complementary DNA fragment containing the wild type or mutant LINC01050 fragment and the 3’untranslated region (UTR) of SPZ1 was subcloned downstream of the luciferase gene within the pmirGLO luciferase reporter vector. HEK293T cells were co-transfected with the LINC01050-WT, LINC01050-MUT, SPZ1-WT, or SPZ1 MUT reporter plasmids individually and together with the miR-7161-3p mimics or NC mimics. At 48 h post-transfection, the firefly and Renilla luciferase activities were measured using a Dual-Luciferase Reporter Assay System (Promega). The ratio of the firefly luciferase to Renilla activity was calculated for each sample.
The Animal Experimental Ethics Committee of Wenzhou Medical University approved all the animal experiments. Four-to-six-week-old male athymic nude mice were purchased from the Zhejiang Charles River Laboratory Animal Co.Ltd. (Zhejiang, China). The nude mice were randomly grouped (n = 5 per treatment group) and were injected subcutaneously with 5 × 106 KATO III cells transduced with lentiviral shNC or shLINC01050. The tumor length and width were measured using a vernier caliper every 5 days. The tumor volume (mm3) was calculated as follows: 0.5 × length × (width).2 The mice were euthanized, and the tumors were isolated on day 25. For the tumor metastasis experiment, 5 × 106 BGC823 cells transduced with lentiviral shNC or shLINC01050 were suspended in 200 μL PBS and were injected into the tail vein of the athymic nude mice (n = 5 per group). The body weight of the mice was measured every 3 days. Forty-three days later, the mice were euthanized, and the lung metastases were evaluated.
Immunohistochemistry
The streptavidin-biotin peroxidase complex method was used for immunohistochemical staining of the formalin-fixed, paraffin-embedded tissue sections. The tissue samples were dehydrated, embedded with paraffin, and cut into 4-μm-thick sections. The paraffin sections were dewaxed by dimethylbenzene and rehydrated by a gradually reduced concentration of ethanol. Antigen retrieval was performed by heating the dewaxed and dehydrated sections in an antigen retrieval solution containing 10 mM EDTA (pH 8.0) using a pressure cooker. Endogenous HRP activity was blocked with 3% H2O2. The primary antibodies were goat anti-human Ki-67 (ab16667, Abcam, USA; 1: 250 dilution) and mouse anti-human PCNA (ab29, Abcam, USA; 1: 10000 dilution). The sections were observed and photographed with an optical microscope (Leica, Wetzlar, Germany).
Northern blot
LINC01050 northern blot was performed using a Roche DIG Northern Starter Kit (Roche, Switzerland) according to the manufacturer’s instructions. A total of 15 μg of RNA from each sample was subjected to formaldehyde gel electrophoresis and transferred to a HyBond N+ Nylon membrane (Amersham). The PCR primers used to generate the northern blot probe were 5′-GGAAGCAGCAAGGTCAATAC-3′ (forward) and 5′-AACAGGCT CCTCAAACAACT-3′ (reverse).
RNA-fluorescence in situ hybridization (RNA-FISH)
RNA-FISH was performed to determine the subcellular location of LINC01050. The LINC01050 anti-sense FISH probe Mix was designed and synthesized by RiboBio (RiboBio Biotechnology, Guangzhou, China). According to the manufacturer’s protocol, the in situ hybridization was carried out with a fluorescent in situ hybridization (FISH) Kit. The fluorescence signals were scanned using the confocal laser microscope system (Leica, Wetzlar, Germany).
Chromatin immunoprecipitation (ChIP) assays
The ChIP assay was performed using a ChIP assay kit (Millipore, Billerica, MA) according to the manufacturer’s protocol. Briefly, the KATO III cells were cross-linked with 1% formaldehyde for 10 min at 37 °C and were sonicated to shear the DNA to lengths between 200 and 1000 bp. Then, 10 μL of the supernatant was used as the input, and the remaining was diluted in the ChIP dilution buffer with protease inhibitor. The chromatin solution was incubated at 4 °C overnight with protein A + G magnetic beads coated with the anti-c-Myc antibody (3 μg) or IgG. A magnetic beads/antibody/histone complex was washed using a complex wash buffer, and the bead-bound immunocomplexes were eluted using an elution buffer. To reverse the histone-DNA crosslinks, the immune complexes were combined with 20 μL of 5 M NaCl, heated for 4 h at 65 °C, treated with proteinase K, and incubated at 45 °C for 1 h. The bound DNA fragments were purified and subjected to PCR using the specific primers. The specific PCR primers are listed in Additional file
1: Table S3.
RNA immunoprecipitation (RIP)
RIP was performed using the EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s protocol. An AGO2 antibody (Abcam, ab32381) and the corresponding IgG were used for the immunoprecipitation. The co-precipitated RNAs were detected by real-time PCR.
RNA pull-down assay
The LINC01050 biotin-labeled RNA probes were transcribed with a biotin RNA labeling mix (Roche, Switzerland) and T7 RNA polymerase (Roche, Switzerland) and treated with RNase-free DNase I (Promega, Madison, WI, USA) in vitro. After purification, the biotinylated RNAs were incubated with the cell lysate at 37 °C for 1 h. M-280 Streptavidin magnetic beads (Invitrogen, USA) were added to the KATO III cell lysate, and the mix was incubated at room temperature for 30 min with rotation. A Western blot assay was used to determine the AGO2 protein expression.
For the RNA-RNA pull-down assay, the cell lysate was incubated with the biotin-labeled LINC01050 using a Biotin RNA Labeling System at 4 °C overnight. The M-280 beads were added later. The co-immunoprecipitated RNAs were washed with buffers and purified. The purified miR-7161-3p RNAs were analyzed by qRT-PCR.
Statistical analysis
All the experimental data are expressed as the mean ± standard deviation (SD). The statistical analyses were performed using SPSS 21.0 software (SPSS Inc., Chicago, IL, USA) or GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA, USA). Statistically significant differences were calculated using an independent sample t-test. P < 0.05 indicated a statistically significant difference.
Discussion
Recently, growing evidence has revealed that the newly discovered lncRNAs play pivotal roles in human diseases, especially cancer. The oncogene c-Myc is often deregulated in human cancers and contributes to tumor progression [
26]. As a transcriptional factor, c-Myc is involved in many biological processes, such as metabolism, cell growth, cell cycle regulation, and apoptosis [
27]. It targets many protein-coding genes. In addition, many lncRNAs are newly-proven downstream targets of c-Myc [
28‐
33], and play essential roles in cancer cell proliferation and tumorigenesis [
33‐
35]. Lu et al. reported the c-Myc-targeted lncRNA DANCR was overexpressed in various tumor types and promoted cancer cell proliferation [
35]. In addition, the c-Myc-induced lncRNA, LncRNA-MIF, plays an important role in c-Myc-mediated aerobic glycolysis [
33]. Cao et al. likewise identified a novel c-Myc-induced lncRNA, LAST, which interacts with CNBP to promote the stability of
CCND1 mRNA [
36].
In this study, we identified LINC01050 as a novel c-Myc-activated lncRNA that functions as a molecular sponge to absorb cytosolic miR-7161-3p, thereby reducing the miR-7161-3p-mediated translational repression of
SPZ1, which contributes to GC progression (Fig.
8h). However, no significant association between c-Myc and LINC01050 expression in the context of GC was identified based on TCGA data (Additional file
2: Fig. S13), suggesting that LINC01050 expression may be regulated in a more complex manner, not just by c-Myc alone. To date, the biological function and expression pattern of LINC01050 in cancer have not been unraveled. We found that LINC01050 was up-regulated in GC tissues and cell lines, and its high expression in GC patients was positively correlated with a poor prognosis. Furthermore, LINC01050 overexpression promoted GC cell proliferation, migration, invasion, and EMT in vitro and tumor growth in vivo. At the same time, its knockdown inhibited GC cell proliferation, migration, invasion, and EMT in vitro, as well as tumor growth and metastasis in vivo
. These results indicate that LINC01050 might play a vital role in GC progression.
The ceRNA theory indicates that lncRNAs function as sponges for miRNAs and thereby regulate the expression of coding genes [
37,
38]. For example, the novel lncRNA, MCM3AP-AS1, promotes the growth of hepatocellular carcinoma by acting as a ceRNA for miR-194-5p [
39]. In addition, the lncRNA LINC01234 promotes the growth of gastric cancer by acting as a ceRNA for miR-204-5p [
40]. We found that LINC01050 localized to the cytoplasm and nucleus, suggesting that it may partly function as an endogenous miRNA sponge. Bioinformatics analyses and luciferase reporter assays revealed that miR-7161-3p was a target of LINC01050. miR-7161-3p overexpression was found to inhibit GC cell growth, migration, invasion, and EMT. Furthermore, rescue experiments revealed that overexpression of miR-7161-3p partly reverse the growth-promoting effect induced by LINC01050, indicating that LINC01050 promots GC progression, at least in part, through the suppression of miR-7161-3p activity.
In regulation effected by the ceRNA network, miRNA targets are integral. Using the TargetScan database, we identified SPZ1 as a potential miR-7161-3p target. SPZ1 is up-regulated in various human cancers and functions as a tumor promoter [
41,
42]. For example, Wang LT and colleagues found that SPZ1 promoted EMT and metastasis in liver cancer [
21,
43], specifically by trans-activating TWIST1, which encodes a master regulator of EMT [
43]. In addition, SPZ1 homodimers activate TWIST1 expression and are acetylated by TIP60 to form a heterodimeric SPZ1-TWIST1 complex, which promotes EMT and initiates tumor metastasis [
44]. Moreover, SPZ1 overexpression in breast cancer promotes drug-resistance and metastases [
45]. To confirm that SPZ1 is a direct target of miR-7161-3p, we conducted luciferase reporter assays and verified that miR-7161-3p targetsits 3′UTR. Overexpression of miR-7161-3p in GC cells suppressed SPZ1 mRNA and protein expression. In addition, we found that LINC01050 regulates
SPZ1 expression through its interaction with miR-7161-3p. There was also a positive correlation between LINC01050 and
SPZ1 expression in GC tissues, and analysis of TCGA data revealed that
SPZ1 mRNA was significantly up-regulated in GC. Finally, knockdown of
SPZ1 by siRNA inhibited GC cell proliferation, migration, invasion, and EMT. Together, these results suggest that LINC01050 modulates GC cell proliferation, migration, invasion, and EMT by regulating the miR-7161-3p/
SPZ1 axis.
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