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DNA Methylation-Mediated Silencing of Regenerating Protein 1 Alpha (REG1A) Affects Gastric Cancer Prognosis

Background Gastric cancer (GC) is one of the most common cause of cancer-related deaths. The clinical trials still lack the effective methods to treat or monitor the disease progression. In this research, the biological function and the underlying molecular mechanism of regenerating protein 1 alpha (REG1A) in GC were investigated. Material/Methods Gene expression omnibus (GEO), KMplot datasets and GC tissue microarray (n=164) were used to analyze the expression of REG1A and related patient prognoses in GC. Transwell matrigel assay, flow cytometry analysis and CCK8 cell viability assay were performed to detect the biological functions of REG1A. Western blotting and real-time PCR were used to detect the REG1A expression and PI3K/Akt related signaling. Results It was found that the expression of REG1A was significantly downregulated in GC and closely related with clinicopathological findings or patient prognoses. REG1A overexpression could suppress the invasion, cell viability and promote the apoptosis of GC cells. Moreover, we found that the epigenetic methylation suppressed the expression level of REG1A in GC, and REG1A overexpression could suppress the phosphorylation of Akt or GSK3β signaling. Conclusions Taken together, REG1A regulates cell invasion, apoptosis and viability in GC through activating PI3K/Akt-GSK3β signaling. REG1A may serve as a promising therapeutic strategy for GC.


Background
Gastric cancer (GC) is the fifth most common malignancy and the third leading cause of cancer-related death worldwide, and half of the world total cases occur at Eastern Asia. GC has 2 distinct morphologic subtypes: gastric intestinal type adenocarcinoma and diffuse gastric adenocarcinoma [1,2]. Gastric intestinal type adenocarcinoma is often associated with intestinal metaplasia and Helicobacter pylori infection, and diffuse gastric adenocarcinoma is more often seen in female and young individuals [3]. It has been known that GC included frequent inactivating mutations in cell adhesion and chromatin remodeling genes in addition to TP53 mutations [4,5]. Although some essential factors for resolution were identified in recent years, but the clinical trials still lack the effective methods to treat or monitor the disease progression [6][7][8][9].
Regenerating protein 1 alpha (REG1A) is a secreted protein containing 166 amino acids and belongs to the calcium dependent lectins superfamily. In humans, 4 different subtypes of REG genes have been known: REG1A, REG1B, REG3A and REG4. REG1A was first identified by screening a cDNA library of regenerating rat pancreatic islet cells and has been primarily implicated in the regeneration of pancreatic b cells and in the amelioration of diabetes mellitus [10][11][12][13]. Previous studies have reported that REG1A play important roles in various cancers, such as: advanced thoracic esophageal squamous cell carcinoma [14], cutaneous melanoma [15], lung cancer [16], hepatocellular carcinoma (HCC) [17], colorectal cancer [18][19][20], breast cancer [21], bladder cancer [22]. Furthermore, REG1A was also reported to be related with the anti-apoptotic effect and angiogenesis in GC [23][24][25], but other biological functions, like the invasion and proliferation of GC cells, have not been investigated yet. In this research, we will deeply investigate the biological functions of REG1A and uncover related underlying mechanism.
Here, we found that the expression of REG1A was significantly downregulated in GC. REG1A expression was closely correlated with clinicopathological findings and patient prognoses. DNA methylation-mediated silencing of REG1A could regulate the invasion, apoptosis and viability of GC cells through phosphatidylinositol 3 kinase (PI3K)/Akt-GSK3b signaling.

Cell culture
Human GC cell lines, including AGS, BGC-823, HGC-27, MGC-803, MKN-45 and SGC-7901 cells, and human gastric epithelial cell line GES-1 were purchased from Cell Bank of the Chinese Academy of Sciences. RPMI-1640 medium supplemented with 10% (v/v) fetal calf serum (FCS) and 1% antibiotics was used here. Cells were placed at 37°C in a humidified incubator with 5% CO 2 .

Clinical samples
A human tissue microarray containing 164 cases of GC samples was obtained from the Department of Bone Tumor, Yantai Mountain Hospital. All human materials were obtained with informed consent, and protocols were approved by the Ethics Review Committee of the World Health Organization Collaborating Center for Research in Human Production.

Immunohistochemical staining
Human tissue microarray was deparaffinized and rehydrated for histopathological evaluation. For immunohistochemical staining, the section was incubated with 0.3% hydrogen peroxide/ phosphate-buffered saline for 30 min and blocked with 10% BSA (Sangon). Slides were first incubated using the antibody for REG1A (Abcam) at 4°C overnight with optimal dilution, labeled by HRP second antibody (Abcam) at room temperature for 1 h. Then, the section was treated with DAB substrate liquid (Thermo) and counterstained by hematoxylin. The section was observed and photographed with a microscope (Carl Zeiss). The final expression of REG1A was designated as low or high expression group as follows: 0-35% positive staining was low expression and more than 36% positive staining was high expression.

Quantitative real-time PCR
Total RNA was extracted by Trizol (Takara), and reversely transcribed by PrimeScript RT-PCR kit (Perfect Real-Time). Quantitative real-time PCR analyses were performed with SYBR Premix Ex Taq (Takara) on a 7500 real-time PCR system (Applied Biosystems).

Western blotting
Cells were lysed in lysis buffer and proteins were separated by SDS-PAGE under reducing condition. The membrane was blocked in phosphate-buffered saline/Tween-20 containing 5% BSA. Then, the antibodies for REG1A (Abcam), phospho-Akt (Cell Signaling), total-Akt (Cell Signaling), phospho-GSK3b (Cell Signaling), total-GSK3b (Cell Signaling), GAPDH (Sigma), and species-specific secondary antibodies were used to incubate the membrane separately. The secondary antibodies were detected using the Odyssey imaging system (LI-COR).

Invasion assays
Cells were detached and resuspended in serum-free RPMI-1640 medium. We placed 2×10 4 cells in 0.1 ml in matrigel (BD)-coated inserts (Millipore) seated on the 24-well plate, then we added 5% FBS RPMI-1640 medium to the bottom chamber. Cells were incubated at 37°C. After 48 h, filters were fixed and stained with 0.1% (w/v) Crystal Violet. Non-invading cells were removed, and invading cells were counted under a microscope at 400×. Three grids per field were counted and the experiments were repeated twice.

Apoptosis assays
We placed 5×10 5 cells/well in 12-well plates and serum starved for 48 h at 37°C. After 48 h, adherent cells were detached harvested in complete RPMI-1640 medium. After centrifuging at 1000 rpm for 5 min, the cells were washed with 1×PBS and stained with 100 μl binding buffer containing 3 μl Annexin V and 3 μl propidium iodide (PI). After incubating at room temperature for 15 min, the cells were analyzed by flow cytometry (BD).

DAC and TSA treatment
Cells were treatment with 5 or 10 μM of 5-aza-2'-deoxycytidine (DAC, Sigma-Aldrich) or 300 or 600 nM Trichostatin A (TSA, Selleckchem) for 3 days and drug in medium were replaced every 24 h. Control cells were incubated with the same volume of DMSO.

Statistical analysis
Data are presented as means ± standard error of the mean (SEM). Survival time was analyzed by Kaplan-Meier method. The Pearson's c 2 test and t test were used for comparisons between groups.

REG1A is downregulated in GC tissues and is closely related with vascular embolism, tumor size, and patient prognoses
To investigate the expression of REG1A in GC, we first analyzed the microarray data from the Gene expression omnibus (GEO). The serial number of the GEO dataset used in this research was GSE 62944. We downloaded the raw data of GSE62944 from  The Cancer Genome Atlas (TCGA). It was shown that the infection of H. pylori virus was not found in these patients. The dataset showed that the expression level of REG1A was significantly downregulated in GC tissues compared with normal gastric tissues ( Figure 1A). By analyzing data from KMplot, we found that REG1A expression was closely related with patient prognoses. High REG1A expression was associated with improved overall survival (OS) (P<0.001) and disease-free survival (DFS) (P=0.002) ( Figure 1B, 1C).
To further investigate the clinical significance of REG1A in GC, we used a GC tissue microarray containing 164 samples. We performed immunohistochemistry to detect the positive staining of REG1A in 164 cases GC tissue microarray. We found that the expression of REG1A was significantly downregulated in 79.01%, up-regulated in 12.04%, and no change in 8.95% of GC patients, compared with normal gastric tissues ( Figure 1D). REG1A expression was closely related with tumor size, vascular embolism, differentiation, patient smoking history, and TNM stage (Table 1). We also found that high REG1A expression was associated with improved OS (P=0.002) and DFS (P=0.036) of these patients ( Figure 1E, 1F).

Overexpression of REG1A suppresses the cell viability of GC cells
To investigate the biological functions of REG1A in GC, we detected the relative mRNA expression level of REG1A in 6 GC cell lines and human gastric epithelial cell line GES-1. As shown in Figure 2A

Overexpression of REG1A suppresses the invasion and promotes the apoptosis of GC cells
We further investigated the invasion and apoptosis of MGC-803 and BGC-823 cells after REG1A overexpression. By transwell matrigel invasion assay, we found that REG1A overexpression suppressed the invasiveness of MGC-803 and BGC-823 cells after 48 h ( Figure 3A, 3B). After the cells were serum starved for 48 h, we found that REG1A overexpression significantly promoted the apoptosis of GC cells by flow cytometry analysis.
The results showed that the apoptosis rate was obviously increased by REG1A overexpression in MGC-803 and BGC-823 cells ( Figure 3C, 3D).
REG1A overexpression has no effect on gastric epithelial cells, but REG1A knockdown increases the proliferation of gastric epithelial cells We used a non-cancer cell line-gastric epithelial cell line (GES-1 cell) to further investigate the biological functions of REG1A. We overexpressed the REG1A gene in GES-1 cells ( Figure 4A). By performing transwell matrigel invasion assay and flow cytometry analysis, we found that REG1A overexpression had no effects on the invasion and apoptosis of GES-1 cells ( Figure  4C). Additionally, by CCK8 cell viability assay, we found that the cell viability of GES-1 cells was not affected by REG1A overexpression ( Figure 4D). Furthermore, we knocked down REG1A in GES-1 cells by siRNA ( Figure 4B). We found that REG1A knockdown increased the proliferation of GES-1 cells at 48 and 72 h, but it had no effects on the invasion and apoptosis of GES-1 cells (Figure 4E, 4F).
DNA methylation silences REG1A expression in GC, and REG1A overexpression suppresses the phosphorylation of Akt and GSK3b signaling We further investigated why REG1A was downregulated in GC. Using 5-aza-2'-deoxycytidine (DAC), a specific methyltransferase inhibitor, and Trichostatin A (TSA), a histone deacetylase inhibitor, we found that methylation occurred in all of the 4 tested GC cell lines, and histone acetylation existed in 1 of them. These data indicate that epigenetic methylation suppresses the expression level of REG1A in GC ( Figure 5A-5D).
To uncover the underlying mechanism of REG1A in GC, we detected PI3K/Akt-related signaling pathway in REG1Aoverexpressed MGC-803 cells and control cells by Western blotting. Interestingly, we found that the phosphorylation of Akt and GSK3b was significantly suppressed by the overexpression of REG1A ( Figure 5E, 5F), suggesting that the regulation of REG1A in GC cell invasion, apoptosis and viability are mainly dependent on PI3K/Akt-GSK3b signaling.

Discussion
In previous studies, REG1A was shown to act as a factor to reduce epithelial apoptosis in inflammation [26]. In recent years, it was found that REG1A plays important roles in the tumorigenesis of various types of human cancer [16][17][18][19][20][21][22]. But some biological functions and related mechanism of REG1A in GC remain unclear. In our research, the exact role of REG1A in GC 5837 was investigated. By analyzing GEO and KMplot datasets, we found that the expression of REG1A was obviously downregulated in GC. High REG1A expression was associated with improved OS and DFS of GC patients. We further confirmed that REG1A expression was closely related with tumor size, vascular embolism, differentiation, patient smoking history, TNM stage, and prognoses of patients by using GC tissue microarray, showing that REG1A may play an important role in the development of GC.
Further, it has been reported that REG1A plays an important role in the anti-apoptotic effect and angiogenesis in GC [23][24][25]. In the present study, we revealed that REG1A also affected other biological functions of GC cells. We found that the invasion and viability of GC cells were suppressed, and the apoptosis of GC cells was supported by REG1A overexpression. REG1A overexpression was proved to have no effects on the invasion, apoptosis or cell viability of non-cancer cell line-gastric epithelial cells. These data indicate that REG1A regulates the invasion, apoptosis, and viability of GC cells, meaning it is involved in the progression of GC.
It is generally considered that, during carcinogenesis processes, epigenetic modifications of the genome, including DNA methylation and histone modification, are established as a consequence of interactions between cellular lineage and    5842 environmental input. However, increasing evidence indicates that epigenetic changes are responsible for initiation, proliferation, invasion, and metastasis of tumor cells by affecting the tumor microenvironment [27,28]. In the present study, by treatment of DAC and TSA in GC cell lines, we proved that epigenetic DNA methylation suppresses the expression level of REG1A in GC. Additionally, we conclude that REG1A was epigenetically silenced by the aberrant hypermethylation of CpG islands in promoter in GC. The epigenetic regulation needs further in-depth study.
PI3K/Akt is a very important signaling pathway involved in various cancers, including GC. GSK3b is a multi-substrate target protein kinase and also an important downstream regulator of the PI3K/Akt pathway. The overexpression of REG1A is correlated with decreased p-AKT and p-GSK3b expression, resulting in the suppression of GC cells invasion and viability and increasing apoptosis. Our results show that the PI3K/Akt-GSK3b signaling pathway participates in the regulatory effects of REG1A on GC cells.

Conclusions
We found that REG1A overexpression suppressed GC cells invasion, viability, and promoted apoptosis. Epigenetic methylation suppressed the expression level of REG1A in GC. REG1A exerts its functional roles in GC mainly through the PI3K/Akt and GSK3b signaling pathways. REG1A may be used as a potential therapeutic strategy for GC in future.

Conflicts of interest
None.