Hypermethylated long noncoding RNA MEG3 promotes the progression of gastric cancer

LncRNA MEG3 and ATP4B were downregulated in gastric cancer tissues

Heat map analysis showed that MEG3 was downregulated among the top 20 differentially expressed lncRNAs in GC and adjacent normal tissues (Figure 1A). Additionally, ATP4B expression was reduced among the top 20 differentially expressed mRNAs between these tissues (Figure 1B). Expression values are represented in shades of red and green, which indicate high expression levels and low expression levels, respectively. All the results suggested that MEG3 and ATP4B were downregulated in GC patients.

Figure 1. Heat map analysis and target gene prediction. (A) Heat map analysis showed that MEG3 was downregulated among the top 20 differentially expressed lncRNAs in GC tissues and adjacent normal tissues. (B) Heat map analysis showed that the expression of ATP4B was reduced among the top 20 differentially expressed mRNAs between GC tissues and adjacent normal tissues. (C) The network of the relationships between the top twelve differentially expressed genes in lncRNA and the top thirty differentially expressed genes in mRNA. (D) MEG3 and ATP4B are highlighted in a Venn analysis of miRNA associated with gastric cancer from the TCGA database.

WGCNA was used to detect gene expression profiles, and the gene coexpression modules were constructed. Coexpression clusters for normal and tumor tissues were detected by hierarchical cluster analysis (Supplementary Figure 1), and each vertical line represents one gene. Supplementary Figure 1 shows the module details of normal tissues. Supplementary Figure 2 displays the size of each module for GC tissues, and we can deduce the correlation between the sample trait and module eigengene. A total of 9 gene modules are shown (Supplementary Figure 2); the genes marked red are hub genes in GC tissues. These genes are ADAMTS2, LMX1A, MYH11, TAGLN, ZG16, ATP4B, RBP2, MAGEA4, and IBSP for red, blue, pink, purple, yellow, black, brown, green and magenta modules, respectively. The Y-axis value represents the significance of the gene in each module, and the X-axis value represents the correlation between the module and gene. The ATP4B module in black was chosen for the following analysis.

A network was constructed of the relationships between the top twelve differentially expressed genes in the lncRNA analysis and the top thirty differentially expressed genes in the mRNA analysis, indicating that they were in contact with each other (Figure 1C). The Venn diagram analysis showed that miR-181a-5p was most correlated with MEG3 and ATP4B from the TCGA data (Figure 1D). Among them, miR-181a-5p has the strongest relationship with GC, so it is the target of further study.

Clinical significance of lncRNA MEG3 expression in GC tissues

We further explored the relationship between MEG3 expression and clinicopathological factors. The MEG3 expression level was correlated with TNM staging, lymph node metastasis and tumor size (Supplementary Table 1) but not with age, sex, extrathyroidal extension, or multicentricity of the clinical pathological characteristics examined.

MEG3 was hypermethylated and expressed at low levels in GC tissues and cells

LncRNA MEG3 was found to be expressed at low levels in thirty GC samples compared to normal tissues (Figure 2A, P<0.01). LncRNA MEG3 was also found to be downregulated in BGC-823, SGC-7910, MGC-803 and AGS GC cell lines, compared to the GES 1 normal cell line (Figure 2B). The MSP results showed that lncRNA MEG3 was hypermethylated in tumor tissues compared with normal tissues (Figure 2C, P<0.01). Hypermethylation of lncRNA MEG3 was also detected in BGC-823, SGC-7910, MGC-803 and AGS GC cell lines compared to the GES 1 normal cell line (Figure 2D). Since these changes were most significant in MGC-803 cells, this cell line was selected for the following analysis.

Figure 2. The expression and methylation status of target factors in gastric cancer and gastric cancer cell lines. (A) LncRNA MEG3 was found to be expressed at low levels in thirty GC samples compared to normal tissues. **P<0.01, compared with 30 matched normal tissues. (B) LncRNA MEG3 was also found to be downregulated in BGC-823, SGC-7910, MGC-803 and AGS GC cell lines compared to the GES 1 normal cell line. *P<0.05, **P<0.01, compared with the GES 1 normal cell line. (C) MSP results showed that lncRNA MEG3 was hypermethylated in tumor tissues compared with normal tissues. **P<0.01, compared with 30 matched normal tissues. (D) Hypermethylation of lncRNA MEG3 was also detected in BGC-823, SGC-7910, MGC-803 and AGS GC cell lines compared to the GES 1 normal cell line. *P<0.05, **P<0.01, compared with the GES 1 normal cell line. (E) MiR-181a-5p was upregulated in tumor tissues compared with their corresponding normal tissues. **P<0.01, compared with 30 matched normal tissues. (F) QRT-PCR results showed higher expression of miR-181a-5p in BGC-823, SGC-7910, MGC-803 and AGS GC cell lines compared to the GES 1 normal cell line. *P<0.05, **P<0.01, compared with the GES 1 normal cell line. (G) QRT-PCR results showed lower expression of ATP4B in tumor tissues compared to normal tissues. **P<0.01, compared with 30 matched normal tissues. (H) QRT-PCR results showed higher expression of ATP4B in BGC-823, SGC-7910, MGC-803 and AGS GC cell lines compared to the GES 1 normal cell line. *P<0.05, **P<0.01, compared with the GES 1 normal cell line.

The expression of ATP4B and miR-181a-5p in GC tissues and cells

MiR-181a-5p was found to be upregulated in tumor tissues compared with their corresponding normal tissues (Figure 2E, P<0.01). QRT-PCR results exhibited higher expression of miR-181a-5p in BGC-823, SGC-7910, MGC-803 and AGS GC cell lines than in the GES 1 normal cell line (Figure 2F). These findings suggested that miR-181a-5p was upregulated in GC patients. In addition, qRT-PCR results showed lower expression of ATP4B in tumor tissues compared to normal tissues (Figure 2G, P<0.01). ATP4B was also downregulated in BGC-823, SGC-7910, MGC-803 and AGS GC cell lines compared to the GES 1 normal cell line (Figure 2H, P<0.01).

The target verification of lncRNA MEG3, miR-181a-5p and ATP4B

The luciferase reporter assay showed that the luciferase activity in the MEG3-wt group mimics + miR was lower than that in the NC group (Figure 3C, P<0.01). RNA pull-down experiments showed that MEG3 enrichment levels in the bio-miR-181a-5p, DMSO + bio-miR-181a-5p and 5-Aza groups were higher than those in the bio-NC group (Figure 3A, P<0.01). In Figure 3B, linear regression analysis showed that the expression of MEG3 was negatively correlated with the expression of miR-181a-5p. As shown in Figure 3G, the expression of miR-181a-5p in the MEG3 overexpression group was significantly lower than that in the NC group (P<0.01). In addition, the enrichment level of ATP4B in the inhibitor - miR group was higher than that that in the mimics - miR group (Figure 3E, P<0.05). Linear regression analysis by qRT-PCR showed that the expression of ATP4B was negatively correlated with miR-181a-5p (Figure 3F), and the expression level of ATP4B in the miR-181a-5p overexpression group was significantly lower than that in the NC group (Figure 3D). All the results showed that ATP4B and miR-181a-5p were negatively regulated.

Figure 3. Verification of the target relationship between the three target genes. (A) The RNA pull-down test showed a low level of MEG3 enrichment in the bio-NC group than in the bio-miR-181a-5p, DMSO + 5-Aza and 5-Aza treatment groups. **P<0.01, compared with the bio-NC group. (B) A negative correlation between the expression of MEG3 and miR-181a-5p was detected by linear regression analysis after qRT-PCR detection of 30 tissue specimens. (C) Luciferase reporter assay showed lower luciferase activity in the mimic-miR + lncRNA MEG3-wt group than in the mimic-miR + lncRNA MEG3-mut group. *P<0.05, **P<0.01, compared with the NC group. (D) The expression of ATP4B was verified by qRT-PCR after the overexpression of miR-181a-5p. **P<0.01, compared with the NC group. (E) The RNA pull-down test showed a higher level of ATP4B enrichment in the inhibitor-miR group than in the mimic-miR group. *P<0.05, compared with the NC group. (F) qRT-PCR detection showed a negative relationship between the expression of ATP4B and miR-181a-5p. (G) The expression of miR-181a-5p was verified by qRT-PCR after the overexpression of MEG3. *P<0.05, compared with the NC group. (H) The expression level of each treatment group MEG3. *P<0.05, compared with the NC group.

The restored expression of MEG3 inhibited the progression of GC cells

We verified the expression levels of MEG3 in each treatment group and found significant increases in the overexpression group and the 5-Aza treatment group (Figure 3H). The number of clones was significantly reduced in the lncRNA MEG3 overexpression group and the 5-Aza group compared with the NC and sh-MEG3 + 5-Aza groups (Figure 4A). The apoptosis rate of MGC-803 cells transfected with MEG3 or 5-Aza dramatically increased compared with the NC and sh-MEG3 + 5-Aza groups (Figure 4B, P<0.05). Wound healing and transwell assays showed significantly reduced migration and invasion ability of cells in the MEG3 and 5-Aza groups relative to the NC and sh-MEG3 + 5-Aza groups (Figure 4C–4D). When we combined all the data, we concluded that MEG3 served as a tumor inhibitor gene suppressing the migration, invasion, and proliferation of GC cells while promoting apoptosis.

Figure 4. Restored expression of MEG3 inhibits the development of gastric cancer cells. (A) The number of clones was significantly reduced in the lncRNA MEG3-overexpressing group and the 5-Aza group compared with the NC group. *P<0.05, **P<0.01, compared with the NC group. #P<0.05, compared with the 5-Aza group. (B) The apoptosis rate of MGC-803 cells transfected with MEG3 or 5-Aza dramatically increased compared with the NC group. *P<0.05, **P<0.01, compared with the NC group. #P<0.05, compared with the 5-Aza group. (C–D) Wound healing and transwell assays showed significantly reduced cell migration and invasion ability in the MEG3 and 5-Aza groups compared to the control group. *P<0.05, **P<0.01, compared with the NC group. #P<0.05, compared with the 5-Aza group.

Overexpression of MEG3 inhibits tumor growth in vivo

To investigate the effect of MEG3 on tumor growth, we transfected MGC-803 cells with MEG3 to observe tumor size and volume. The results showed that tumor weight and volume in the MEG3 group were lower than those in the Con group (Figure 5A–5C, P<0.01). Next, we studied the expression of ATP4B and miR-181a-5p after MEG3 transfection. The results showed that the expression of ATP4B was increased in the MEG3 group, while the expression of miR-181a-5p was decreased (Figure 5D–5E, P<0.01). Therefore, we demonstrated a negative correlation between MEG3 and miR-181a-5p and found that upregulation of MEG3 in vivo can inhibit tumor growth.

Figure 5. Overexpression of MEG3 in vivo inhibits tumor growth. (A–C) Tumor xenograft results showed that tumor weight and volume were decreased in the MEG3 group compared with the Con group. *P<0.05, **P<0.01, compared with Con group. (D) QRT-PCR results illustrated that ATP4B expression decreased while miR-181a-5p expression increased in the MEG3 group compared with the Con group. (E) Western blotting results illustrated that ATP4B expression increased in the MEG3 group compared with the Con group. *P<0.05, **P<0.01, compared with Con group.

Clinical samples

According to previous studies, we made reference to the selection of case samples, and the selected tissue samples were chosen by blind selection of two pathologists for inclusion in this study. Finally, we determined 30 patients as the sample number of this study [25, 33, 34]. A total of 30 paired tumor and normal tissues were obtained from GC patients who were undergoing treatment at the First Hospital of Jilin University (Jilin, China). The corresponding noncancerous tissues were at least 5 cm away from the tumor edge. The specimens were snap-frozen in liquid nitrogen and stored at a temperature of -80°C for molecular analyses. All subjects gave informed consent for obtaining the study materials. The study protocol was agreed by the Clinical Research Ethics Committee of the First Hospital of Jilin University (Jilin, China).

Cell culture

Four human gastric cancer cell lines (BGC-823, SGC-7910, MGC-803 and AGS) and a normal human gastric mucosa cell line (GES 1) were received from BeNa Culture Collection (Beijing, China). Cells were kept at 37°C in a humid atmosphere with 5% CO₂. BGC-823 cells were cultured in Roswell Park Memorial Institute (RPMI) medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% FBS (Invitrogen, CA, USA). SGC-7910 cells were cultured in Roswell Park Memorial Institute 1640 (RPMI1640) medium (Sigma-Aldrich, St. Louis, MO, USA) with 10% FBS (Invitrogen, CA, USA). MGC-803 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM – 214.5 g/L glucose) containing 10% FBS (Invitrogen, Carlsbad, CA, USA). AGS cells were cultured in Nutrient Mixture F-12 (F-12) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FBS (Invitrogen). GES 1 cells were cultured in RPMI medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% FBS (Invitrogen).

Methylation-specific PCR (MSP)

Bisulfite-converted DNA (~50 ng) was amplified in a one-step MSP reaction with 0.2 μM of each deoxynucleotide triphosphate and primer (both methylated and unmethylated forward and reverse primers) using 1 U Maxima® Hot Start Taq DNA polymerase (Thermo Fisher Scientific, Inc.). The respective PCR conditions were as follows: one cycle at 98°C for 30 seconds (initial denaturation); 35 cycles at 98°C for 10 seconds (denaturation), 62°C for 30 seconds (annealing) and 72°C for 30 seconds (extension); and one cycle at 72°C for 7 minutes (final extension). MSP validity was hindered by the amplitude of CpG-methylated human genomic DNA acting as the negative control; however, for the non-methylation-specific PCR, human genomic DNA that was not converted by bisulfite was utilized as the positive control. PCR procedures were performed with a BIOER™ Thermal Cycler 9500 (Hangzhou Bioer Technology Co., Ltd., Binjiang, China). The percentage of methylated reference (PMR) was calculated as the fraction of the methylated reference [(methylated molecules in a sample)/(total DNA molecules in a sample)]/[(methylated molecules in SssI-treated DNA)/(total DNA molecules in SssI-treated DNA)] [35]. All of the primers involved were purchased from AuGCT Biotechnologies (Beijing, China), and primer sequences are shown in Supplementary Table 2.

Cell transfection

To enhance the exogenous expression of MEG3, the full length human MEG3 was synthesized by PCR according to the GenBank gene sequence (NR_002766) and inserted into the pcDNA3.1 empty plasmid (GenePharma, Shanghai, China); Control (pcDNA3.1 vector) was used for comparison [14]. To reduce the expression of MEG3, GenePharma synthesized shRNA (MEG3 shRNA) against MEG3. The sequence of the MEG3 shRNA is as follows: 5′-GAGAGGTTGTTTCACTGGTATCTATTG CA-3′. MGC-803 cells were seeded in 96-well plates and cultured to 60% confluency for transfection. Transfection was performed using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. The concentrations of pcDNA3.1-MEG3 and MEG3 shRNA for transfections were 100 nM.

Demethylation with 5-Aza-dC treatment

Cells were seeded at a density of 1 × 10⁶ cells/ml. After 24 hours, cells were treated with 1 μM 5-Aza-dC (Sigma-Aldrich, St. Louis, MO, USA) and an equivalent concentration of vehicle (dimethyl sulfoxide) for 5 days and then were harvested for DNA and RNA extractions.

Luciferase reporter assay

The 3′UTR fragment of lncRNA MEG3 was amplified and replicated into the PmeI and XbaI sites of the pmirGLO vector (Promega, Madison, WI, USA). The mutant MEG3 3′UTR fragment was also developed using site-directed mutagenesis. According to reporter assays, cells were formed in 24-well plates and transfected with MEG3-wt or MEG3-mut type luciferase reporters and then cotransfected with miR-181a-5p mimics or mimics control. Each assay was performed three times. Treated cells were analyzed for luciferase activity with the Dual-Glo Luciferase Assay System (Promega, WI, USA) and a MicroLumatPlus LB96V luminometer (Berthold, USA) after 48 hours.

RNA pull-down test

Biotinylated miR-181a-5p, biotinylated mutant, and biotinylated NC were synthesized by GenePharma (Shanghai, China). The biotinylated miRNA was transfected into MGC-803 cells. The cell lysates were incubated with M-280 streptavidin magnetic beads (Invitrogen, USA). RT–qPCR was employed to probe the levels of MEG3.

QRT-PCR

RNA from cells or tissue samples was separated with the mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA) and reverse transcribed to cDNA with the First Strand cDNA Synthesis Kit (Fermentas, Burlington, Canada). These cDNAs were transferred to qPCR with the SYBR premix ExTaq kit (TaKaRa, Dalian, China) to probe the expression of lncRNA MEG3, ATP4B or miR-129-5p. For lncRNA MEG3 and ATP4B expression, paired primers (forward and reverse) were utilized, and glyceraldehyde phosphate dehydrogenase (GAPDH) acted as the endogenous reference gene. For miR-181a-5p expression, paired primers (forward and reverse) were utilized with U6 snRNA for normalization. All of the reactions were carried out with an Applied Rotor-Gene 6000 Real Time machine. The relative expression was calculated using the $2^{-\Delta \Delta C_t}$ method [36]. All of the primers involved were purchased from AuGCT Biotechnologies (Beijing, China) and are shown in Supplementary Table 2.

Transwell invasion assay

Eight-micrometer pore polycarbonate membranes (BD Bioscience) coated with 25 μg Matrigel® (BD Biosciences) were applied in transwell chambers. A total of 5 × 10⁴ cells in a medium containing 0.5-1% FBS was placed in the upper chamber, while 5-10% FBS medium was placed in the lower chamber. Non-migrated cells were removed with a cotton swab while migrated cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet after 24 hours. The total number of migrated cells was counted and photographed with a Nikon Eclipse E600 microscope (Nikon Instruments, Japan).

Clone formation assay

A total of 5 × 10³ cells were seeded into six-well culture plates and replenished every 3 days with complete medium. Cells were subsequently fixed with methanol and 0.5% crystal violet. Visible colonies were manually counted in randomly selected fields with a Nikon Eclipse E600 microscope (Nikon Instruments, Melville, New York, USA). The clone formation rate (CFR) was computed according to the following formula: CFR = clone counts/seeded cell counts ×100%. The experiment was repeated 3 times.

Flow cytometry analysis

Transfected MGC-803 cells were washed and resuspended. Immunofluorescence staining was performed at 4°C with the construction of the Annexin V-FITC Apoptosis Staining/Detection kit (Cambridge, MA, USA). After cleaning with FACS buffer, cells were delivered to conduct multichannel analysis by a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA) and were then analyzed with Cell-Quest 3.3 software (BD Biosciences).

Wound healing assay

Transfected cells were seeded into 6-well plates at 1 × 10⁵ cells/l. A 10 μl Eppendorf tip was used to scratch the center of the plate. The cell movement was recorded at 0, 24 and 48 hours by Image-Pro Plus 6.0 software. The migration ability was computed as follows: (prime scratch width ‒ current scratch width)/prime scratch width. The experiment was repeated 3 times.

Tumor xenograft

Experimental animals (4-week-old female BALB/c nude mice) were purchased from the Animal Center of the First Hospital of Jilin University (Jilin, China). For epigenetic therapy, MGC-803 cells (1× 10⁶/200 μl PBS) were subcutaneously inoculated into the right flank of BALB/c nude mice. When the average tumor size reached approximately 100 mm³, the animals were separated into 2 groups. The epigenetic therapy used in this study was the sh-MEG3 group (5 mg/kg) and the sh-Con group. Tumor diameters were measured with a Vernier caliper, and mice were evaluated every five days.

Weighted gene coexpression network analysis

The coexpression network module is identified using the average RPM value in R and the WGCNA package (v3.4.1) [37]. Genes with a lower mean RPM coefficient of variation (CV < 1) in all sample types (cell/tissue type, different latitude sections or developmental stages) were excluded, and the remaining genes were used for analysis. The average relative RPM value summarizes the expression patterns of each module. The module characteristic gene (ME) value for each module is calculated, which summarizes the expression profile of a given module as the first principal component. The intermodule similarity is calculated as the PCC between the ME values. The signed KME algorithm was used to calculate the PCC between each gene RPM expression level and the module ME, and the ME-based gene connectivity (kME) was calculated, which is a quantitative factor indicating the correlation intensity of individual genes in each module. Package WGCNA_3.4.1 was used in this study to screen the hub gene in each gene module [38].

Statistical analysis

Statistical analysis was conducted with GraphPad Prism 6.0 Software (GraphPad Inc., La Jolla, CA, USA). All of the experiments were repeated at least three times. Differences in expression among groups were tested using one-way analysis of variance (ANOVA). The chi-square test was used to stratify and compare clinical samples. A value of P < 0.05 was regarded as statistically significant.