Long non‐coding RNA LINC01225 promotes proliferation, invasion and migration of gastric cancer via Wnt/β‐catenin signalling pathway

Abstract Emerging evidence has classified the aberrant expression of long non‐coding RNAs (lncRNAs) as a basic signature of various malignancies including gastric cancer (GC). LINC01225 has been shown to act as a hepatocellular carcinoma‐related gene, with its expression pattern and biological function not clarified in GC. Here, we verified that LINC01225 was up‐regulated in tumour tissues and plasma of GC. Analysis with clinicopathological information suggested that up‐regulation of LINC01225 was associated with advanced disease and poorer overall survival. Receiver operating characteristic (ROC) analysis showed that plasma LINC01225 had a moderate accuracy for diagnosis of GC. In addition, knockdown of LINC01225 led to retardation of cell proliferation, invasion and migration, and overexpression of LINC01225 showed the opposite effects. Mechanistic investigations showed that LINC01225 silencing inhibited epithelial‐mesenchymal transition (EMT) process and attenuated Wnt/β‐catenin signalling of GC. Furthermore, ectopic expression of Wnt1 or suppression of GSK‐3β abolished the si‐LINC01225‐mediated suppression against EMT, thereby promoting cell proliferation, invasion and migration of GC. In conclusion, LINC01225 promotes the progression of GC through Wnt/β‐catenin signalling pathway, and it may serve as a potential target or strategy for diagnosis or treatment of GC.

non-coding RNAs (ncRNAs). 3 Long non-coding RNAs (lncRNAs) are a class of ncRNAs that transcribe longer than 200 nucleotides, with limited or no protein-coding capacity. 4 Once considered as transcriptional noise, lncRNAs have recently been implicated in a wide range of biological processes including normal tissue development, regulation of cellular pluripotency, modulation of cell proliferation and apoptosis. Specifically, lncRNAs are emerging as key players in genetics and pathogenesis of cancer and their dysfunction is closely associated with cancer development, progression and metastasis including GC. 5,6 By acting as oncogenes or tumour suppressors, lncRNAs may contribute to GC tumorigenesis and progression. For example, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) functions as a competing endogenous RNA (ceRNA) to attenuate the inhibitory effect of miR-23b-3p on autophagy-related protein 12 (ATG12) and then leads to chemo-induced autophagy and chemoresistance in GC. 7 Moreover, long intergenic non-coding RNA (lincRNA) FEZF1 antisense 1 (FEZF1-AS1) represses p21 expression to promote GC proliferation through lysine-specific demethylase 1(LSD1)-mediated H3K4me2 demethylation. 8 In contrast, lncRNA GAS5 enhances G1 cell cycle arrest via binding to YBX1 to regulate p21 expression in GC. 9 Our previous study showed that lncRNA MT1JP suppressed GC cell proliferation and migration by modulating miR-214-3p/RUNX3 axis. 10 Given that the current understanding on GC-associated lncRNAs may represent only the tip of iceberg, additional wok is still needed to further clarify the functional roles of many other lncRNAs in GC development.
Located at chromosome 1p35.2, LINC01225 is a lincRNA with a length of 2113 base pairs (bps). 11 Wang et al first reported that LINC01225 acted as a hepatocellular carcinoma (HCC)-related ln-cRNA and that its high expression was significantly associated with advanced TNM stage, larger tumour size and positive lymph node metastasis. 11 Specifically, they found that LINC01225 promoted HCC growth and invasion via epidermal growth factor receptor/mitogen-activated protein kinase (EGFR/MAPK) pathway. 11 However, the role of LINC01225 in other cancers including GC is unknown up to now and remains to be elucidated.
In this study, we firstly examined the expression pattern of LINC01225 in tumour tissues and plasma of GC. Our results demonstrated upregulation of LINC01225 in GC to be associated with advanced disease and poorer overall survival. Furthermore, LINC01225 promoted cell proliferation, motility and EMT potential of GC through the Wnt/β-catenin signalling pathway. Collectively, our findings support an oncogenic role of LINC01225 in GC progression and suggest that LINC01225 may be served as a candidate biomarker for diagnosis, staging and outcome prediction of GC.

| Data extraction and analysis
The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database were selected to learn the expression pattern of LINC01225 in GC initially. Raw RNA sequencing data containing 344 gastric adenoma and adenocarcinoma samples and 30 matched non-cancerous samples were obtained from TCGA database. The data were then normalized and analysed with the edgeR package.
Microarray dataset (GSE54129) containing transcriptome data of 111 GC samples and 21 non-cancerous samples was downloaded from GEO database and normalized using Robust Multichip Average (RMA) method. The z-score of log 2 format of normalized data was used for further analysis.

| Patients and specimens
A total of 109 pairs of GC tissues and adjacent non-cancerous tissues were obtained from patients who had undergone radical gastrectomy in the Affiliated People's Hospital of Jiangsu University between 2015 and 2016 for this study. The adjacent normal tissues were obtained from tissues that were located 5 cm away from the edge of GC. All selected GC patients met the following inclusion criteria: (a) patients were newly diagnosed to have GC with definite pathological evidence; (b) no chemoradiotherapies were given before surgery. Plasma samples were collected from each subject, as well as 45 healthy donors included in the same period, and stored at −80°C before use. Tumour stage was evaluated according to the eighth TNM staging of the International Union against Cancer (UICC)/American Joint Committee on Cancer (AJCC) system. 12 This study was approved by the Institutional Ethical Committee of Jiangsu University, and signed informed consents were obtained from all patients. were cultured with Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific). All media were supplemented with 10% foetal bovine serum (FBS; Gibco), 100 U/mL penicillin and 100 μg/ mL streptomycin (Gibco) in humidified air at 37°C with 5% CO 2 .

| RNA isolation and reverse transcriptionquantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from tissues, plasma and differ-  and Wnt1 overexpression plasmids were also purchased from GenePharm. For transfection, the cells were grown in a 12-well plate until confluence at 60%-80% and were transfected with the indicated molecules with Lipofectamine 2000 (Thermo Fisher Scientific). For pharmacological inhibition of GSK-3β activity, the cells were treated with 20 mmol/L lithium chloride (LiCl; Sigma-Aldrich) as previously described. 13 Equivalent dose of NaCl served as vehicle.

| Cell counting, colony formation and apoptosis
For proliferation assay, GC cells were seeded into 24-well plates at a density of 1 × 10 4 cells/well. The cells were collected and counted everyday for 6 days. The results were plotted as cell growth curves. For colony formation assay, the cells were placed in 6-well plates (1000 cells/ well) and were cultured for 10 days. The colonies were then washed, fixed and stained with 0.5% crystal violet. Visible colonies were counted and photographed. For cell apoptosis assay, the indicated cells were stained with Annexin V-FITC/PI cell apoptosis detection kit (BD Pharmingen) and analysed with a flow cytometer (BD FACSCalibur).
Experiments were carried out in triplicate independently.

| Migration and invasion assay
Migration and invasion assay was performed in transwell chambers without or with Matrigel-coated membranes, respectively. Briefly, GC cells were seeded with serum-free medium into the upper chambers at 5 × 10 4 cells/well, and the bottom chambers contained medium with 10% FBS. After culture for 24 hours, cells on the upper surface were removed with a cotton swab and cells on the lower surface were stained and counted under a microscope.

| Wound-healing assays
Indicated cells were plated to confluence in 6-well plates. Streaks across the plate were created in the monolayer with a pipette tip, followed by three washes in serum-free medium. The cells were then cultured for 24 hours with medium containing 1% FBS. To ensure documentation of the same region, the wells were marked across the wounded area. Progression of migration was observed and photographed at 0 and 24 hours after wounding. The distance between the two edges of the scratch was measured and calculated.

| Immunohistochemistry
Briefly, sections were deparaffinized, rehydrated and incubated in sodium citrate buffer for antigen retrieval. Slides were photographed under a microscope.

| Statistical analysis
All statistical analyses were conducted with SPSS 24.0 software (IBM, SPSS). Student's t tests or one-way analysis of variance (ANOVA) was performed to determine the significance of differences between different groups. The associations between LINC01225 and the clinicopathological features were analysed by the Pearson chisquared test. Survival curves according to LINC01225 expression were generated using Kaplan-Meier method, and their difference was evaluated by log-rank test. Receiver operating characteristic (ROC) curve was established to evaluate the diagnostic value of LINC01225 for GC. A two-sided P value < .05 was considered statistically significant.

| Up-regulation of LINC01225 correlated with advanced disease and worse outcome of GC
To understand the expression pattern of LINC01225 in GC, we com- with lower expression of LINC01225 in TCGA database ( Figure 1C).
To validate these findings, we then detected LINC01225 expression levels in a cohort of 109 paired GC tissues and adjacent non-cancerous tissues by RT-qPCR. As shown in Figure 1D, the expression levels of LINC01225 were significantly higher in tumour tissues than those in adjacent non-cancerous tissues. We further found that 65 cases displayed at least twofold increase in GC tissues compared with the paired non-cancerous tissues ( Figure 1E). Importantly, up-regulation of LINC01225 was significantly correlated with advanced TNM stage, increased invasion depth and positive lymphatic metastasis (  Figure 1F). In addition, smoke was significantly associated with increased LINC01225 level (Table 1).Consistent with the TCGA findings, we also observed a worse overall survival for patients with high LINC01225 expression ( Figure 1G).
To further explore the potential utility of LINC01225 for GC diagnosis, we detected the expression of LINC01225 in plasma samples from 45 randomly selected GC patients and paired healthy donors.
The stability of circulating lncRNA has been well documented in previous studies. 10,11 Herein, we found that the expression levels of plasma LINC01225 were significantly higher in GC patients than those in healthy controls ( Figure 1H). Up-regulation of plasma LINC01225 was significantly associated with advanced TNM stage and positive lymphatic metastasis, respectively ( Figure 1J). Finally, plasma LINC01225 had an area under the ROC curve (AUC) of 0.755 (95% CI, 0.655-0.854, P < .001) for diagnosis of GC ( Figure 1I). These findings suggest that LINC01225 expression in tissue or plasma may be a candidate predictor for GC diagnosis and staging assessment.

| LINC01225 knockdown inhibited proliferation, migration and invasion of GC cells
To investigate the roles of LINC00978 in GC, we firstly profiled its ex-  Figure 2I). Taken together, our findings suggest that LINC01225 may enhance GC progression by promoting proliferation, invasion and migration and by inhibiting of apoptosis.

| LINC01225 knockdown suppressed epithelialmesenchymal transition (EMT) of GC
Since EMT plays a crucial role in cancer progression by conferring an invasive phenotype, 14  The bold values mean P < .05, that indicates the difference reach statistical signifcance. and mesenchymal markers in SGC-7901 and BGC-823 cells was examined upon LINC01225 knockdown. As shown in Figure 3A

| Wnt/β-catenin signalling contributed to LINC01225-mediated EMT process
We then asked the mechanisms by which LINC01225 regulated EMT process of GC cells. Previous work has revealed that many growth factors, such as Wnt, transforming growth factor-β (TGF-β) and Notch, are engaged to trigger and complete an EMT process. 15 Specifically, a close interplay between Wnt signalling and cadherinmediated cell adhesion has been supported by profound evidence. 16 Previous studies have also provided evidence that many lncRNAs such as LINC01133, LINC01197 and SLCO4A1-AS1 may affect EMT through direct or indirect regulation of Wnt signalling. 17 Here, we examined Wnt-related genes by LINC01225 knockdown and found that Wnt signalling was distinctly suppressed, as represented by decreased mRNA and protein expression of Wnt1 and β-catenin( Figure 3B,E). These data suggest that Wnt signalling acts downstream of LINC01225.
Activation of Wnt signalling can induce GSK-3β inhibition, reversal of β-catenin phosphorylation and thereby promote β-catenin accumulation and nuclear relocation. 20 We observed that si-LINC01225-induced decrease in Wnt1 did not alter the expression or Ser9 phosphorylation of GSK-3β ( Figure 3E), indicating that LINC01225 silencing mainly affected the activity of GSK-3β. This is in line with the reports that Wnt-dependent GSK-3β inhibition relies on the direct interaction of GSK-3β with the scaffold protein Axin and the associated protein adenomatous polyposis coli (APC) rather than the expression or serine phosphorylation of GSK-3β. 21,22 We further determined the activity of GSK-3β by measuring the phosphorylation of β-catenin and found that LINC01225 silencing restored β-catenin phosphorylation at Ser37/Thr41/Ser45 and destabilized β-catenin ( Figure 3E). Furthermore, we tested whether overexpression of Wnt1 had similar effects as inhibition of GSK-3β exerted. As expected, ectopic expression of Wnt1 also reversed si-LINC01225-mediated EMT suppression in SGC-7901 cells ( Figure S2A-C).Therefore, these findings support that Wnt/β-catenin signalling contributes to LINC01225-mediated EMT process.

| Inhibition of GSK-3β counteracted the tumour suppressive effect by LINC01225 silencing
Since Wnt signalling acted downstream of LINC01225 to promote EMT process, we then asked whether Wnt signalling was involved in LINC01225-mediated phenotype change in GC. To test this, LINC01225-silencing SGC7901 cells were co-transfected with si-GSK-3β or treated with LiCl and cell proliferation and motility were investigated. As expected, si-LINC01225-mediated inhibition of cell proliferation, migration and invasion was completely reversed by si-GSK-3β or LiCl ( Figure 4A-C).

| LINC01225 silencing suppressed GC tumorigenesis in vivo
The effect of LINC01225 on GC tumorigenesis in vivo was determined by using a subcutaneous xenograft tumour model. As shown in Figure 5A,  can mediate TGF-β-induced EMT and is shown to promote metastasis in various cancers. 27 Similarly, Chen et al indicated that MANCR (MALAT2-activated lncRNA) contributes to GC migration by inducing EMT via the MEK/ERK pathway. 28 In this study, we found that LINC01225 knockdown reduced N-cadherin, vimentin and MMP-7 expression and increased E-cadherin expression, suggesting that LINC01225 crucially regulated the EMT process of GC cells.

Consistently, EMT-inducing transcription factors including Snail and
Slug were also down-regulated by LINC01225 silencing. For example, MALAT1 can bind to CTNNB1 promoter region and recruit methyltransferase to promote CTNNB1 promoter methylation, thereby inhibiting CTNNB1 and modulating the Wnt signalling pathway. 34 Besides, LINC01197 restrains Wnt/β-catenin signalling by disrupting the interaction between β-catenin and TCF4 in pancreatic ductal adenocarcinoma. 17 In addition, LINC01133 directly targets miR-106a-3p and in turn inhibits GC metastasis through inactivating the APC/Wnt/β-catenin pathway. 19 Therefore, ongoing work is strongly needed to determine the localization of LINC01225 and to clarify the DNA binding potential, the ceRNA potential or the protein binding potential of LINC01225 with regard to Wnt signalling. This is also the focus of our future research.
In conclusion, our results showed that the expression of LINC01225 was increased in GC and its high expression correlated with the disease malignancy. LINC01225 promoted proliferation, migration, invasion and EMT process of GC cells through Wnt/βcatenin signalling pathway. Our findings not only provide novel evidence for LINC01225-mediated progression of GC but also suggest a potential target or strategy for diagnosis or treatment of GC. had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest.