Background
Gastric cancer (GC) is the fourth most common cancer, the third leading cause of cancer-associated mortality in males and the fifth leading cause of cancer-associated mortality in females worldwide. Almost half of the GC patients were Chinese, most of them were at an advanced stage with lymph node or distal metastasis when diagnosed [
1,
2]. Although a number of GC treatments have been developed in recent years, the prognosis of GC patients remain poor [
3]. Thus, it is important to elucidate the new mechanisms of GC development to identify new ways to prevent GC progression.
Wilms’ tumour gene on the X chromosome (WTX), also known as AMER1 or FAM123B, was the first tumour suppressor gene located on the X chromosome discovered in the Wilms’ tumour [
4‐
6]. Because of its unique location, a “one hit” somatic inactivation that targets the single allele in males or the active X allele in females can cause WTX gene inactivation [
7]. The loss of WTX is also associated with the tumorigenesis of nephroblastoma [
8]. The results of mechanistic studies have shown that WTX inhibits the WNT/β-catenin signaling pathway activation by binding β-catenin [
9]. Furthermore, WTX antagonizes WNT/beta-catenin signaling by promoting β-catenin ubiquitination and degradation [
10]. WTX is also able to enhance p53 acetylation by interacting with CBP/p300 protein [
11]. However, as a candidate tumour-suppressor gene, the expression and functions of WTX have not been fully elucidated in other tumours.
In our previous study, WTX was shown to be generally lost in GC, and the stomach was identified as another important target organ of the WTX gene [
12]. The WTX gene has a 16.3% [
13,
14] to 30% [
15] mutation rate in Wilms’ tumour, which drives WTX lost. However, WTX mutations and high methylation levels of WTX promoters are rare among GC patients [
16,
17], suggesting that there may be another mechanism responsible for WTX gene silencing in GC patients. Aberrant microRNA (miRNA) expression is another important factor involved in regulating gene silencing. miRNAs can regulate gene expression at the post-transcriptional level by binding to the 3′-untranslated region (3’UTR) of target mRNAs. Interestingly, miRNAs have been reported to regulate the transcription of approximately 60% of genes [
18] that participate in several bioprocesses, including organ development, cell proliferation, apoptosis [
19,
20], and EMT [
21,
22]. However, whether the loss of WTX in GC is associated with miRNAs remains unknown.
The phosphoinositide-3 kinase (PI3K)–protein kinase (PKB/AKT)–mammalian target of rapamycin (mTOR) pathway plays a crucial role in regulating multiple cellular functions and inducing pathological processes, including the cell cycle, proliferation, quiescence, tumorigenesis, and progression [
23‐
25]. Deregulation of PI3K/AKT/mTOR pathway has been frequently demonstrated in GC and is closely associated with GC tumorigenesis and prognosis [
26‐
29]. However, a relationship between WTX and the PI3K/AKT/mTOR pathway has yet to be reported. Therefore, to better understand the function of WTX and identify new markers for targeted GC therapy, in the present study, in vitro and in vivo assays and bioinformatics analyses were performed to elucidate the function and mechanisms of WTX loss and the relationship between WTX and the PI3K/AKT/mTOR pathway in GC progression.
Methods
Tissue specimens and cell culture
One hundred sixty-one cases of GC and matched adjacent normal gastric tissue samples were obtained from patients at Nanfang Hospital, Southern Medical University. No chemotherapy or radiation therapy was administered prior surgery. Prior patient consent and approval were obtained from the Institutional Research Ethics Committee. GC cell lines (GES-1, SGC7901, AGS, BGC803, MGC803, MGC823, BGC823, MKN28, and MKN45) were obtained from the Cell Bank of Chinese Academy of Medical Science (Shanghai, China). These cells were cultured in PPMI-1640 medium containing 10% fetal bovine serum (Invitrogen Life Technology) and incubated at 37 °C under a humidified atmosphere with 5% CO2. All cell lines were routinely tested for mycoplasma, the results of which were negative.
Overexpression and knockdown cell lines
A lentivirus expression vector (LV-W-Puro) harbouring the entire WTX CDS region was synthesized by Invitrogen Co. (Shanghai, China). WTX shRNAs fragments were synthesized by Genechem Co. (Shanghai, China), verified and used to establish lentivirus expression vector (LV-shW-Puro). MiR-20a-5p mimics and inhibitor lentiviruses (LV-20 am-puro and LV-20ai-puro) were constructed by GenePhama (Shanghai, china). Target cells (2 × 105) were infected with 1 × 106 lentivirus transducing units in the presence of polybrene (1 μg/ml) and subsequently selected with puromycin (2 μg/ml) for ~ 5 days to establish the stable overexpression and knockdown cell lines.
Cell proliferation, colony formation, migration, and wound healing assays
Cell proliferation was assessed using the cell counting kit-8 (CCK8) assay. Briefly, 2 × 103 cells were seeded into each well of 96-well plates in triplicate. After incubating for the indicated time, 5 μl of CCK-8 buffer (DOJINDO, Japan) was added to each well, and the cells were incubated for 1 h at 37 °C. Then, the absorbance of each well was measured at 450 nm using a Microplate Autoreader (Bio-Rad, Hercules, CA, USA).
For colony formation assays, cells were plated in 6-well plates (300 cells/well) and cultured for approximately 2 weeks until the appearance of cell colonies. The colonies were fixed and stained with hematoxylin, and those containing more than 50 cells were counted for further statistical analysis. Each experiment was performed thrice.
For cell migration assay, 1 × 105 cells were seeded into Transwell chambers (BD Biosciences, San Jose, CA, USA) under the same treatment conditions described above and incubated for 48–72 h. Cells that had migrated through the filter were stained with haematoxylin and counted in five fields per insert for further statistical analysis.
For wound healing assays, pretreated cells were seeded into 6-well plates and scratched with a pipette tip after 24 h. Then, after the abraded, floating cells were removed with PBS, the adhered cells were continuously cultured for another 48 h, with images taken every 8 h. Each experiment was performed thrice.
Immunohistochemistry (IHC), in situ hybridization (ISH), and scoring
Paraffin-embedded sections were dewaxed from xylene, rehydrated in a graded ethanol series to water, and used for IHC staining according to a previously described protocol [
12]. The samples were incubated with primary antibodies against the following proteins: WTX (diluted 1:200, Abcam, Cambridge, UK), Ki-67 (diluted 1:500, Abcam, Cambridge, UK), p-AKT and p-mTOR (diluted 1:200, Cell Signaling Technology, Danvers, MA).
ISH assay were performed under RNase-free conditions. The miR-20-5p probe and ISH kit were purchased from Boster Co. (Wuhan, China). The slides were stepwise dewaxed, rehydrated in a graded ethanol series to water, and refixed following a previously described protocol [
12]. Then, they were incubated in 50 μg/ml Proteinase K, prehybridize for 4 h at 60 °C, 1.5 μg/ml probe incubated 18 h at 60 °C in series; following rinsing 2×, 1×, and 0.1 × SSC; blocking, anti-digoxin-biotin, SABC-POD, streptavidin-HRP, DAB substation, and hematoxylin counterstain in sequence to detect the positive signal.
The results were scored as previously described as a sum of the staining intensity and percentage of positive tumour cells [
12]. Briefly, the staining intensity was scaled from 0 to 3. The percentage of cells showing positive staining was scored from 0 to 4. The final staining score (0–12) was calculated as the sum of the intensity and percentage scores and was then adapted to 4-point IRS scores as follows: 0–1 (−), 2–3 (+), 4–8 (+ +) and 9–12 (+ + +). Finally, we set (+) as the WTX expression cut off point, with (−) and (+) ~ (+ + +) indicating negative and positive expression, respectively. The clinical pathological significance of the IHC data were analyzed by Chi-square analysis. IHC staining and scoring were performed in a blind manner.
Immunofluorescence (IF)
The cells were cultured in dishes and then fixed with 4% paraformaldehyde at 4 °C for 15 min. Then, after being blocked with normal goat serum for 30 min, the cells were incubated with primary antibodies against WTX (1:200) and PI3K (1200) at 4 °C overnight, after which they were incubated with Alexa Fluor 488-conjugated Affinipure goat Anti-mouse IgG (H + L) and 594-conjugated goat anti-rabbit IgG(H + L) (Proteintech, Chicago, IL) for 2 h at room temperature. Subsequently, DAPI was used to stain cell nuclei. Then, the stained cells were mounted with 80% glycerol/PBS for subsequent examination by a laser-scanning confocal microscope (FV1000, Olympus, Japan) using FV10-ASW 4.0 viewer software (Olympus, Japan).
Immunoblotting
The proteins were eluted with denaturation buffer; separated by SDS-PAGE, and transferred to PVDF membranes (Pierce Biotechnology, Rockford, IL, USA). Subsequently, the menbranes were blocked in milk before being incubated in primary antibodies (WTX, diluted 1:500, Abcam; PI3K, p-PI3K, AKT, p-AKT, mTOR, p-mTOR, P70S6K, 4E-BP1, diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA; GAPDH, diluted 1:2000, Proteintech, Rosemont, IL, USA) and the secondary antibodies. Finally, the proteins were visualized using an enhanced chemiluminescence detection system (Amersham Biosciences Europe, Germany) according to the manufacturer’s instructions. All experiments were repeated at least three times.
RNA extraction and quantitative reverse transcription PCR (RT-PCR)
Total RNA was extracted from tissues and cells lines with RNAiso-Plus (TAKARA, Dalian, China). Single-stranded cDNA was then synthesized from 1 μg extracted mRNA using RT-PCR cDNA synthesis kit (TAKARA, Dalian, China) according to the manufacturer’s instructions. RT-PCR was performed using an Applied Biosystems 7500 Sequence Detection system with iQ™ SYBR green supermix (Bio-Rad Laboratories, Hercules, CA, USA). Primers were prepared as described previously [
12]. Thermal cycling conditions were as follows: 95 °C 10 min for 1 cycle; 95 °C 5 s, 60 °C 30 s, and 72 °C 34 s for 40 cycles followed by the melting curve stage. The relative expression of WTX and miR-20a-5p were evaluated based on the threshold cycle (Ct), which was calculated as 2
−ΔΔCT. All of the samples were analysed in three independent experiments, each with four technical replicates performed in each assay.
Subcutaneous and orthotopic xenograft tumour mouse model
Subcutaneous tumour models were established in 4-week-old BALB/c nude mice (nu/nu) (the Animal Center of Southern Medical University, Guangzhou, China) by subcutaneously injecting 100 μl of a cell suspension (1 × 10
6 cells) into the right inguinal region of each nude mouse. The tumour volumes were measured every 2 days starting 10 days after injection. Tumour diameter was measured using a slide caliper, and the volume was calculated using the following formula: tumour volume (mm
3) = (L × W
2)/2 [
30]. After 30 days of observation, the mice were sacrificed to harvest the tumours for farther analysis.
Orthotopic GC mouse models were established in 4-week-old BALB/c nude mice. After anesthetizing the mice with ketamine (70 μg/kg), a small incision was made in the abdomen and to reveal the stomach. Then, 100 μl of a cell suspension (1 × 106 cells) was injected into the muscle tissue of the stomach. Then, the stomach position was reset, the wound was treated with penicillin, and the abdominal incision was closed. The model mice were sacrificed 2 months after surgery to collect the stomachs, and the tumours were measured and fixed for further histopathological study.
MRNA array
Total RNA was extracted from the AGS.veh and AGS. W cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Subsequently, the total RNA samples were digested with DNaseI, purified using an RNeasy Kit (Qiagen, Hilden, Germany), after which the quantity and quality of the samples were assessed. Then, the RNA was used for cDNA synthesis and the production of biotin-tagged cRNA with a GeneChip IVT Labelling kit (Affymetrix), and the fragmented cRNA with controls were hybridized to each GeneChip array according to the manufacturer’s instructions. Hybridization, data capture, and analysis were performed by CapitalBio Co. (Beijing, China). The Affymetrix Human Genome U133 Plus 2.0 Array (Santa Clara, CA, USA), which contains more than 54,000 probe sets covering more than 47,000 transcripts and variants representing more than 38,500 genes, was used for microarray analysis. The differentially expressed genes were identified using threshold values of ≥2- and ≤ − 2-fold change and an FDR significance level of < 5%. The differentially expressed gene profiles data for the AGS.veh and AGS. W cell lines were deposited on Gene Expression Omnibus (GEO) under the accession code GSE114353.
MiRNA array
Total RNA from GC and matched gastric mucosa tissue was labelled with Hy3 using a miRCURY LNA miRNA Power Labelling kit (Exqion, USA). Human lung fibroblast (HLF) cells was used as controls. MiRNA microarrays (CCDTM-miRNA850-V4p1.4) were provided by the Infectious Disease and Immunogenetics section, Department of Transfusion Medicine, Clinical Center, National Institutes of Health, USA. MiRNA array hybridization and data analysis were performed following the manufacturer’s instructions and a previously described protocol [
31]. The microRNA expression profile data were deposited on GEO under the accession code GSE94882.
Dual-luciferase reporter system analysis
A WTX gene 3’UTR fragment harbouring the miR-20a-5p binding site was PCR amplified from genomic DNA. The sequences of primers used in the present study were previously described [
12]. The WTX gene 3’UTR fragment was specifically mutated using a QuickChange Site-directed Mutagenesis kit (TOYOBO, Shanghai, China). The wild-type or mutated 3’UTR fragments were subcloned into the vector pGL3-control (Promega, Madison, MI, USA) to construct the vectors miR-20a-p-5p-GL3-WTX-3’UTR-WT and miR-20a-p-5p-GL3-WTX-3’UTR-Mut, respectively. Then, the vectors were cotransfected with miR-20a-5p into MGC823 or SGC7901 cells, which were subsequently harvested and analyzed for luciferase activity according to the manufacturer’s instructions. Three independent experiments were performed.
KEGG pathway enrichment analysis
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of DEGs were performed using the web-based tool DAVID 6.8 (
https://david.ncifcrf.gov). The results were visualized in a Bar plot by using
ggplot2 in the R software environment.
Statistical analyses
Each experiment was performed at least 3 times. All data were presented as the means± standard deviation (SD). Statistical analyses were performed using Prism 5 (GraphPad Software, San Diego, CA, USA). Two-tailed Student’s t-test was used to assess the significance of differences between two groups. One-way ANOVA was used to assess the significance of differences among multiple groups. Chi-square test was used to estimate the correlation between WTX expression and clinicopathologic features. Survival curves were analyzed using the Kaplan-Meier method and assessed by log rank testing. P-values< 0.05 were considered to be statistically significant.
Discussion
Tumour suppressor genes are normal genes that slow down cell division, repair DNA mistakes, and contribute to the fidelity of the cell cycle replication process. The inactivation of tumour suppressor genes is an important contributor to GC development and progression [
35]. In our previous study, WTX expression was shown to be generally lost in GC samples, and the stomach was identified as a novel target of WTX [
12]. WTX plays a major role in both organ development and tumour suppression, but its functions have not been well elucidated [
11]. The clinicopathological consequences of WTX loss in GC development were analysed, and the results showed that WTX loss is associated with the poor differentiation, high invasion, and proliferation phenotypes of GC, as well as being associated with increased lymph node metastasis and poor GC patient prognosis. These clinical data suggested that WTX loss is correlated with GC progression and can be used as a prognosis marker in GC patients. The tumour suppressor function of WTX was further confirmed by observation of its ability to prevent GC cell proliferation, migration and invasion in vitro and inhibit GC cell proliferation and metastasis in vivo
. The results of these studies confirmed that WTX is a tumour suppressor gene in GC and that the loss of WTX is at least partially responsible for GC progression.
The mechanism by which WTX functions as a GC tumour-suppressor was analysed for the first time in the present study. Both our gene expression microarray data (GSE114353) and public microarray data (GSE34715) revealed that WTX loss is associated with the aberrant activation of the PI3K/AKT/mTOR pathway, which induces the proliferation and metastasis of GC cells. The regulation of PI3K/AKT/mTOR pathway activity may be one of important mechanisms by which WTX regulates GC progression. The PI3K/AKT/mTOR signaling pathway is known to be frequently activated in GC and plays a crucial role in mediating multiple cellular functions, including cell proliferation, metastasis, and resistance to chemotherapy [
24,
36‐
39]. PI3K/AKT/mTOR functions as an important pathway to regulate the progression of GC [
40‐
43]. The Western blot and IHC staining results further confirmed that WTX negatively regulates PI3K/AKT/mTOR pathway activity and inhibits GC proliferation. For the first time, the results of the present study demonstrated that WTX regulates GC progression by preventing PI3K phosphorylation to inhibit PI3K/AKT/mTOR pathway activation. Our results indicated that the loss of WTX may be one of the causes leading to aberrant PI3K/AKT/mTOR signaling pathway activation in GC, which promotes the progression and metastasis of GC.
Aberrant miRNAs regulation is one of the key reasons leading to target gene silencing. To elucidate the mechanism driving WTX loss, we performed miRNA array analyses of GC and matched normal gastric mucous tissues with low and high WTX expression, respectively, resulting in the identification of miR-20a as a candidate regulator of WTX expression. Subsequently, we performed a bioinformatics analysis based on public miRNA array data and identified miR-20a-5p as a candidate regulator of WTX. It was previously reported that miR-20a is highly expressed in hepatocellular carcinoma [
44], lung cancer [
45], colon cancer [
46], and other tumours [
47,
48], and functions as an oncogene. Furthermore, miR-20a can be used as a biomarker in the diagnosis and prognosis of GC [
49,
50]. The results of the above studies and reports suggests that miR-20a-5p is the most likely candidate capable of regulating WTX expression and GC progression. However, the relationship between miR-20a with WTX in GC has remained uninvestigated. To assess the potential miR-20a-mediated regulation of WTX and the associated mechanism, we performed a luciferase reporter assay and confirmed that WTX is a target of miR-20a-5p. Subsequently, the results of a miR-20a-5p functional analysis showed that miR-20a-5p can inhibit WTX expression and promote GC cell proliferation, invasion, and metastasis both in vitro and in vivo. Thus, the results of our study demonstrated that the aberrant upregulation of miR-20a-5p promotes GC progression by inducing WTX loss. In all GC cell line and public TCGA and GEO data assayed, all KEGG pathway analyses and verification experiments confirmed that miR-20a-5p positively regulates PI3K/AKT pathway activation by inhibiting WTX expression, thereby promoting GC progression.
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