(REG1A) Affects Gastric Cancer Prognosis

LAB/IN VITRO RESEARCH

e-ISSN 1643-3750 © Med Sci Monit, 2017; 23: 5834-5843 DOI: 10.12659/MSM.904706

Received: 2017.04.06 Accepted: 2017.06.05 DNA Methylation-Mediated Silencing of Published: 2017.12.09 Regenerating Protein 1 Alpha (REG1A) Affects Gastric Cancer Prognosis

Authors’ Contribution: BCE 1 Yan-Song Qiu 1 Department of General Surgery, Yantai Mountain Hospital, Yantai, Shandong, Study Design A AE 2 Guang-Jun Liao P.R. China Data Collection B 2 Department of Bone Tumor, Yantai Mountain Hospital, Yantai, Shandong, Statistical Analysis C ABC 2 Ning-Ning Jiang P.R. China Data Interpretation D Manuscript Preparation E Literature Search F Funds Collection G

Corresponding Author: Ning-Ning Jiang, e-mail: [email protected] Source of support: Departmental sources

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 GSK3b signaling. Conclusions: Taken together, REG1A regulates cell invasion, apoptosis and viability in GC through activating PI3K/Akt-GSK3b signaling. REG1A may serve as a promising therapeutic strategy for GC.

MeSH Keywords: Apoptosis • Neoplasm Invasiveness • Stomach Neoplasms

Full-text PDF: https://www.medscimonit.com/abstract/index/idArt/904706

2238 1 5 28

Background fetal calf serum (FCS) and 1% antibiotics was used here. Cells

were placed at 37°C in a humidified incubator with 5% CO2. Gastric cancer (GC) is the fifth most common malignancy and the third leading cause of cancer-related death worldwide, and Clinical samples half of the world total cases occur at Eastern Asia. GC has 2 distinct morphologic subtypes: gastric intestinal type adeno- A human tissue microarray containing 164 cases of GC sam- carcinoma and diffuse gastric adenocarcinoma [1,2]. Gastric ples was obtained from the Department of Bone Tumor, intestinal type adenocarcinoma is often associated with in- Yantai Mountain Hospital. All human materials were obtained testinal metaplasia and Helicobacter pylori infection, and dif- with informed consent, and protocols were approved by the fuse gastric adenocarcinoma is more often seen in female and Ethics Review Committee of the World Health Organization young individuals [3]. It has been known that GC included fre- Collaborating Center for Research in Human Production. quent inactivating mutations in cell adhesion and chromatin remodeling genes in addition to TP53 mutations [4,5]. Although Immunohistochemical staining some essential factors for resolution were identified in recent years, but the clinical trials still lack the effective methods to Human tissue microarray was deparaffinized and rehydrated for treat or monitor the disease progression [6–9]. histopathological evaluation. For immunohistochemical staining, the section was incubated with 0.3% hydrogen peroxide/ Regenerating protein 1 alpha (REG1A) is a secreted protein phosphate-buffered saline for 30 min and blocked with 10% containing 166 amino acids and belongs to the calcium de- BSA (Sangon). Slides were first incubated using the antibody pendent lectins superfamily. In humans, 4 different subtypes for REG1A (Abcam) at 4°C overnight with optimal dilution, la- of REG genes have been known: REG1A, REG1B, REG3A and beled by HRP second antibody (Abcam) at room temperature REG4. REG1A was first identified by screening a cDNA library for 1 h. Then, the section was treated with DAB substrate liquid of regenerating rat pancreatic islet cells and has been primar- (Thermo) and counterstained by hematoxylin. The section was ily implicated in the regeneration of pancreatic b cells and in observed and photographed with a microscope (Carl Zeiss). The the amelioration of diabetes mellitus [10–13]. Previous stud- final expression of REG1A was designated as low or high expres- ies have reported that REG1A play important roles in various sion group as follows: 0–35% positive staining was low expres- cancers, such as: advanced thoracic esophageal squamous cell sion and more than 36% positive staining was high expression. carcinoma [14], cutaneous melanoma [15], lung cancer [16], hepatocellular carcinoma (HCC) [17], colorectal cancer [18–20], Quantitative real-time PCR breast cancer [21], bladder cancer [22]. Furthermore, REG1A was also reported to be related with the anti-apoptotic effect Total RNA was extracted by Trizol (Takara), and reverse- and angiogenesis in GC [23–25], but other biological func- ly transcribed by PrimeScript RT-PCR kit (Perfect Real-Time). tions, like the invasion and proliferation of GC cells, have not Quantitative real-time PCR analyses were performed with been investigated yet. In this research, we will deeply inves- SYBR Premix Ex Taq (Takara) on a 7500 real-time PCR system tigate the biological functions of REG1A and uncover related (Applied Biosystems). underlying mechanism. Western blotting Here, we found that the expression of REG1A was significantly downregulated in GC. REG1A expression was closely corre- Cells were lysed in lysis buffer and proteins were separated lated with clinicopathological findings and patient prognoses. by SDS-PAGE under reducing condition. The membrane was DNA methylation-mediated silencing of REG1A could regulate blocked in phosphate-buffered saline/Tween-20 containing the invasion, apoptosis and viability of GC cells through phos- 5% BSA. Then, the antibodies for REG1A (Abcam), phospho- phatidylinositol 3 kinase (PI3K)/Akt-GSK3b signaling. 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 incu- Material and Methods bate the membrane separately. The secondary antibodies were detected using the Odyssey imaging system (LI-COR). Cell culture Lentivirus production and cell transduction Human GC cell lines, including AGS, BGC-823, HGC-27, MGC-803, MKN-45 and SGC-7901 cells, and human gastric epithelial cell line We cotransfected 293T cells with pEZ-lv105 vector GES-1 were purchased from Cell Bank of the Chinese Academy (GeneCopoeia) by using Lipofectamine 2000 (Invitrogen) for of Sciences. RPMI-1640 medium supplemented with 10% (v/v) virus packaging. Viruses were harvested at 24, 48, and 72 h

A B C 8 1.0 1.0

* vival 6 0.8 0.8

el of REG1A P<0.001 P=0.002 vival 0.6 High expression (n=268) vival sur 0.6 High expression (n=296) 4

ee sur 0.4 ession lev 0.4 erall sur -fr 2 Ov 0.2 0.2

Low expression (n=608) Disease Low expression (n=345) mRNA expr 0.0 0 0.0 Normal Gastric tumor 050 100 150 050 100 150 Time (months) Time (months)

D E F 8.95%

12.04% 1.0 1.0 P=0.002 0.8 High expression (n=109) vival 0.8 P=0.036 vival 0.6 vival sur 0.6 High expression (n=109) Down-regulated

Up-regulated 0.4 ee sur 0.4 erall sur -fr

No change Ov 0.2 Low expression (n=55) 0.2 Low expression (n=55)

79.01% Disease 0.0 0.0 02040 60 80 100 02040860 0 Time (months) Time (months)

Figure 1. The expression of REG1A is downregulated in gastric cancer (GC) tissues and closely related with patient prognoses. (A) REG1A mRNA expression level in GC and normal gastric tissues. We obtained this dataset from TCGA. ** P<0.01. (B, C) KMplot analysis of overall survival (OS) (B, P<0.001) and disease-free survival (DFS) (C, P=0.002) for the expression of REG1A. (D) The expression of REG1A was downregulated in 79.01% of GC tissues. (E, F) Kaplan-Meier analysis of OS (E, P=0.002) and DFS (F, P=0.036) for the expression of REG1A in 164 cases GC tissue microarray.

after transfection. After determining virus titers, 1×105 cells DAC and TSA treatment were infected with 1×106 recombinant lentivirus-transducing units by using 6 μg/ml polybrene (Sigma). Cells were treatment with 5 or 10 μM of 5-aza-2’-deoxycytidine (DAC, Sigma-Aldrich) or 300 or 600 nM Trichostatin A (TSA, Invasion assays Selleckchem) for 3 days and drug in medium were replaced every 24 h. Control cells were incubated with the same volume of DMSO. Cells were detached and resuspended in serum-free RPMI-1640 medium. We placed 2×104 cells in 0.1 ml in matrigel (BD)-coated Statistical analysis inserts (Millipore) seated on the 24-well plate, then we added 5% FBS RPMI-1640 medium to the bottom chamber. Cells were incu- Data are presented as means ± standard error of the mean (SEM). bated at 37°C. After 48 h, filters were fixed and stained with 0.1% Survival time was analyzed by Kaplan-Meier method. The Pearson’s (w/v) Crystal Violet. Non-invading cells were removed, and invad- c2 test and t test were used for comparisons between groups. ing cells were counted under a microscope at 400×. Three grids per field were counted and the experiments were repeated twice. Results Apoptosis assays REG1A is downregulated in GC tissues and is closely We placed 5×105 cells/well in 12-well plates and serum starved related with vascular embolism, tumor size, and patient for 48 h at 37°C. After 48 h, adherent cells were detached har- prognoses vested in complete RPMI-1640 medium. After centrifuging at 1000 rpm for 5 min, the cells were washed with 1×PBS and To investigate the expression of REG1A in GC, we first analyzed stained with 100 μl binding buffer containing 3 μl Annexin V and the microarray data from the Gene expression omnibus (GEO). 3 μl propidium iodide (PI). After incubating at room tempera- The serial number of the GEO dataset used in this research was ture for 15 min, the cells were analyzed by flow cytometry (BD). GSE 62944. We downloaded the raw data of GSE62944 from

The Cancer Genome Atlas (TCGA). It was shown that the in- The results showed that the apoptosis rate was obviously infection of H. pylori virus was not found in these patients. The creased by REG1A overexpression in MGC-803 and BGC-823 dataset showed that the expression level of REG1A was sig- cells (Figure 3C, 3D). nificantly downregulated in GC tissues compared with normal gastric tissues (Figure 1A). By analyzing data from KMplot, we REG1A overexpression has no effect on gastric epithelial found that REG1A expression was closely related with patient cells, but REG1A knockdown increases the proliferation of prognoses. High REG1A expression was associated with im- gastric epithelial cells proved overall survival (OS) (P<0.001) and disease-free survival (DFS) (P=0.002) (Figure 1B, 1C). We used a non-cancer cell line-gastric epithelial cell line (GES- 1 cell) to further investigate the biological functions of REG1A. To further investigate the clinical significance of REG1A in GC, We overexpressed the REG1A gene in GES-1 cells (Figure 4A). we used a GC tissue microarray containing 164 samples. We By performing transwell matrigel invasion assay and flow cy- performed immunohistochemistry to detect the positive stain- tometry analysis, we found that REG1A overexpression had no ing of REG1A in 164 cases GC tissue microarray. We found that effects on the invasion and apoptosis of GES-1 cells (Figure the expression of REG1A was significantly downregulated in 4C). Additionally, by CCK8 cell viability assay, we found that 79.01%, up-regulated in 12.04%, and no change in 8.95% of the cell viability of GES-1 cells was not affected by REG1A over- GC patients, compared with normal gastric tissues (Figure 1D). expression (Figure 4D). REG1A expression was closely related with tumor size, vascular embolism, differentiation, patient smoking history, and Furthermore, we knocked down REG1A in GES-1 cells by siRNA TNM stage (Table 1). We also found that high REG1A expres- (Figure 4B). We found that REG1A knockdown increased the sion was associated with improved OS (P=0.002) and DFS proliferation of GES-1 cells at 48 and 72 h, but it had no effects (P=0.036) of these patients (Figure 1E, 1F). on the invasion and apoptosis of GES-1 cells (Figure 4E, 4F).

Overexpression of REG1A suppresses the cell viability of DNA methylation silences REG1A expression in GC, and GC cells REG1A overexpression suppresses the phosphorylation of Akt and GSK3b signaling To investigate the biological functions of REG1A in GC, we detected the relative mRNA expression level of REG1A in 6 GC We further investigated why REG1A was downregulated in GC. cell lines and human gastric epithelial cell line GES-1. As shown Using 5-aza-2’-deoxycytidine (DAC), a specific methyltransfer- in Figure 2A, we found that the expression level of REG1A in ase inhibitor, and Trichostatin A (TSA), a histone deacetylase MGC-803 and BGC-823 cells was obviously lower than in other inhibitor, we found that methylation occurred in all of the 4 GC cells. Then, we established stable cell lines transduced by tested GC cell lines, and histone acetylation existed in 1 of the lentivirus carrying the REG1A gene, named as Lenti-REG1A, them. These data indicate that epigenetic methylation sup- in MGC-803 and BGC-823 cells. By real-time PCR and Western presses the expression level of REG1A in GC (Figure 5A–5D). blotting analysis, we found that REG1A was overexpressed in MGC-803 (Figure 2B, 2D) and BGC-823 cells (Figure 2C, 2E). To uncover the underlying mechanism of REG1A in GC, we detected PI3K/Akt-related signaling pathway in REG1A- By CCK8 cell viability assay, we investigated the cell viability overexpressed MGC-803 cells and control cells by Western of MGC-803 and BGC-823 cells after REG1A overexpression. blotting. Interestingly, we found that the phosphorylation of We found that the cell viability of MGC-803 and BGC-823 cells Akt and GSK3b was significantly suppressed by the overex- was significantly suppressed by REG1A overexpression at 24, pression of REG1A (Figure 5E, 5F), suggesting that the regula- 48, and 72 h (Figure 2F, 2G). tion of REG1A in GC cell invasion, apoptosis and viability are mainly dependent on PI3K/Akt-GSK3b signaling. Overexpression of REG1A suppresses the invasion and promotes the apoptosis of GC cells Discussion We further investigated the invasion and apoptosis of MGC-803 and BGC-823 cells after REG1A overexpression. By transwell In previous studies, REG1A was shown to act as a factor to re- matrigel invasion assay, we found that REG1A overexpression duce epithelial apoptosis in inflammation [26]. In recent years, suppressed the invasiveness of MGC-803 and BGC-823 cells af- it was found that REG1A plays important roles in the tumori- ter 48 h (Figure 3A, 3B). After the cells were serum starved for genesis of various types of human cancer [16–22]. But some 48 h, we found that REG1A overexpression significantly pro- biological functions and related mechanism of REG1A in GC moted the apoptosis of GC cells by flow cytometry analysis. remain unclear. In our research, the exact role of REG1A in GC

Table 1. Correlation of the clinicopathological findings with REG1A expression. Pearson’sc 2 test was used. The bolding stands for P values with significant differences.

REG1A (n) Variable High Low P

£50 years 39 40 Age 0.102 >50 years 52 33

Female 11 10 Gender 0.912 Male 80 63

Yes 21 45 Smoking history <0.001 No 70 28

Diffuse 26 25 Lauren subtype 0.713 Intestine 65 48

Upper 12 10

Middle 15 12 Location 0.682 Lower 21 21

Remnant stomach 43 30

£5 cm 68 10 Tumor size <0.001 >5 cm 23 63

well 36 1

Differentiation moderate 44 12 <0.001

poor 11 60

Yes 16 44 Vascular embolism <0.001 No 75 29

Yes 13 15 Alcohol habits 0.121 No 78 58

I 65 19

TNM stage II 17 7 <0.001

III 9 47

Pearson’s c2 test was used. The bolding stands for the P-values with significant differences.

was investigated. By analyzing GEO and KMplot datasets, we biological functions of GC cells. We found that the invasion found that the expression of REG1A was obviously downreg- and viability of GC cells were suppressed, and the apoptosis ulated in GC. High REG1A expression was associated with im- of GC cells was supported by REG1A overexpression. REG1A proved OS and DFS of GC patients. We further confirmed that overexpression was proved to have no effects on the inva- REG1A expression was closely related with tumor size, vas- sion, apoptosis or cell viability of non-cancer cell line-gastric cular embolism, differentiation, patient smoking history, TNM epithelial cells. These data indicate that REG1A regulates the stage, and prognoses of patients by using GC tissue microar- invasion, apoptosis, and viability of GC cells, meaning it is in- ray, showing that REG1A may play an important role in the volved in the progression of GC. development of GC. It is generally considered that, during carcinogenesis process- Further, it has been reported that REG1A plays an important role es, epigenetic modifications of the genome, including DNA in the anti-apoptotic effect and angiogenesis in GC [23–25]. In methylation and histone modification, are established as a the present study, we revealed that REG1A also affected other consequence of interactions between cellular lineage and

Indexed in: [Current Contents/Clinical Medicine] [SCI Expanded] [ISI Alerting System] This work is licensed under Creative Common Attribution- [ISI Journals Master List] [Index Medicus/MEDLINE] [EMBASE/Excerpta Medica] NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) 5838 [Chemical Abstracts/CAS] [Index Copernicus] Figure 2. © MedSciMonit,2017;23:5834-5843 DNA methylation-mediated silencingofREG1Aaffects gastriccancerprognosis Qiu Y.-S. etal.: A Relative mRNA level of LMO3 0.02 0.04 0.06 0.08 NonCommercial-NoDerivatives 4.0International (CCBY-NC-ND 4.0) 0 assay analysisofBGC-823cellsinfected withlenti-vectororlenti-REG1Aat0,12,24,48,and72h.** below. ( cells, whichwereinfectedwithlenti-vectororlenti-REG1A.Statistical analysisofREG1Aexpressioninthe2groupsisshown were infectedwithlenti-vectororlenti-REG1A.( human gastricepithelial celllineGES-1. ( Overexpression ofREG1Areduces thecellviabilityofGCcells.(

F GES-1 This work islicensed underCreative CommonAttribution-

F Cell viability (OD 450 nm) ) CCK8assayanalysisofMGC-803cellsinfectedwithlenti-vectoror lenti-REG1Aat0,12,24,48,and72h.( AG 0.3 0.6 0.9 1.2 S 0 BGC- D 823 ** GAPDH REG1A Le V 01 ehicle REG1A/GAPDH densitometry nti-REG1A HGC- 0.2 0.4 0.6 0.8 27 0 Le nti-v MGC- 22 803 ** Le ec nti-v MGC-803 to MGC-803 T MKN- r ime (h) ec 45 ** to 44 r ** Le 7901 nti-REG1A SGC- Le nti-REG1A ** B, C B, 8 ) REG1AmRNAexpressionlevelinMGC-803( D, E D, ** 72 B

) REG1AproteinexpressionlevelinMGC-803( Relative mRNA level of REG1A 0.01 0.02 0.03 5839 0 E GAPDH REG1A/GAPDH densitometry REG1A G Le 0.2 0.4 0.6 0.8 1.0 nti-v 0 A MGC-803 ) ThemRNAexpressionlevelofREG1Ain6GCcelllinesand Le ec

Cell viability (OD 450 nm) Le nti-v to nti-v r ** 0.2 0.4 0.6 0.8 1.0 [Chemical Abstracts/CAS] [ISI Journals MasterList] Indexed in: ec 0 ec BGC-823 to Le to r nti-REG1A r ** Le V 01 ehicle Le nti-REG1A Le nti-REG1A [Current Contents/Clinical Medicine] nti-REG1A 22 C [Index Medicus/MEDLINE] BGC-823 T [Index Copernicus] LAB/IN VITRO RESEARCH ime (h) B ) andBGC-823(

44 Relative mRNA level of REG1A 0.01 0.02 0.03 0 D ** Le ) andBGC-823( 8 nti-v P <0.01. ec BGC-823 to ** 72 [SCI Expanded] r C ** ) GCcells,which [EMBASE/Excerpta Medica] Le nti-REG1A E ) GC G ) CCK8 [ISI Alerting System]

LAB/IN VITRO RESEARCH Figure 3. D A C PI PI 10 10 10 10 10 10 10 10 10 10 NonCommercial-NoDerivatives 4.0International (CCBY-NC-ND 4.0) 0 1 2 3 4 0 1 2 3 4 Annexin Annexin 10 10 Statistical analysisofapoptotic BGC-823cellsinthe2groups isshownatright.** 2 groupsisshownright.( of apoptotic MGC-803cellsinfectedwithlenti-vectororlenti-REG1A.Statistical analysisofapoptotic MGC-803cellsinthe Statistical analysisofinvadedBGC-823cellsinthe2groupsisshown right.Scalebars:10μm.( shown right.Scalebars:10μm.( MGC-803 cellsinfectedwithlenti-vectororlenti-REG1A.Statisticalanalysis ofinvadedMGC-803cellsinthe2groupsis REG1A overexpressionsuppresses theinvasionandsupports theapoptosis ofGCcells. ( 0 0 This work islicensed underCreative CommonAttribution- 10 10 Le V V MGC-803 + + nti-REG1A 1 1 V ehicle Annexin Annexin V V ehicle ehicle 10 10 2 2 V V 10 10 1.15% 3.92% 0.32% 2.30% 3 3 10 10 D 4 4

) Flowcytometryanalysisofapoptotic BGC-823cellsinfected withlenti-vectororlenti-REG1A. Cell number per ﬁeld 120 160 200 40 80 0 MGC-803 BGC-823 B ) Representative photosofinvadedBGC-823cells infectedwithlenti-vectororlenti-REG1A. V ehicle PI PI 10 10 10 10 10 10 10 10 10 10 ** 0 1 2 3 4 0 1 2 3 4 10 10 Le 0 0 nti-REG1A 10 10 1 1 Le Le 5840 Annexin Annexin nti-REG1A nti-REG1A 10 10 B 2 2 V V 10 10 3 3 0.67% 9.45% 2.91% 8.87% Le DNA methylation-mediated silencingofREG1Aaffects gastriccancerprognosis 10 10 BGC-823 [Chemical Abstracts/CAS] [ISI Journals MasterList] Indexed in: nti-REG1A V 4 4 ehicle [Current Contents/Clinical Medicine] P <0.01. A [Index Medicus/MEDLINE] ) Representative photosofinvaded [Index Copernicus] Annexin V+ cell percentage (%) Annexin V+ cell percentage (%) Cell number per ﬁeld 120 160 200 40 80 10 15 10 15 0 0 5 0 5 C ) Flowcytometryanalysis © MedSciMonit,2017;23:5834-5843 V V V ehicle ehicle ehicle [SCI Expanded] ** ** ** [EMBASE/Excerpta Medica] Le Le Le nti-REG1A nti-REG1A nti-REG1A Qiu Y.-S. etal.: [ISI Alerting System]

Qiu Y.-S. et al.: DNA methylation-mediated silencing of REG1A affects gastric cancer prognosis LAB/IN VITRO RESEARCH © Med Sci Monit, 2017; 23: 5834-5843

A B GES-1 GES-1

or A l vect i-REG1 Negative Lenti- Lent contro si-REG1A-1 si-REG1A-2 REG1A REG1A

GAPDH GAPDH

0.8 ** 0.8 ** ry ry ** 0.6 0.6

0.4 0.4

0.2 0.2 REG1A/GAPDH densitomet REG1A/GAPDH densitomet 0 0 Lenti-vector Lenti-REG1A Negative si-REG1A si-REG1A control -1 -2

C GES-1 GES-1 D GES-1

90 9 NS 9 NS Vehicle Lenti-REG1A 60 6 centage (%) 6 OD 450 nm) cell per +

30 V 3 3 ell number per ﬁeld C ell viability ( C 0 Annexin 0 0 Vehicle Lenti-REG1A Vehicle Lenti-REG1A 0122448 72 Time (h)

E GES-1 GES-1 F GES-1 NS NS 120 NS 9 NS 1.2 ** Negative control ** si-REG1A-1 80 6 si-REG1A-2 centage (%) 0.8 *

OD 450 nm) * cell per +

40 V 3 0.4 ell number per ﬁeld C ell viability ( C 0 Annexin 0 0 Negative si-REG1A si-REG1A Negative si-REG1A si-REG1A control -1 -2 control -1 -2 0122448 72 Time (h)

Figure 4. REG1A overexpression has no effect on gastric epithelial cells, while REG1A knockdown promotes the proliferation of gastric epithelial cells. (A) REG1A protein expression level in GES-1 cells, which were infected with lenti-vector or lenti-REG1A. Statistical analysis of REG1A expression in the 2 groups is shown below. (B) REG1A protein expression level in GES-1 cells, which were infected with siRNA of REG1A. Statistical analysis of REG1A expression is shown below. (C) Statistical analysis of invaded or apoptotic GES-1 cells infected with lenti-vector or lenti-REG1A. (D) CCK8 assay analysis of GES-1 cells infected with lenti-vector or lenti-REG1A at 0, 12, 24, 48, and 72 h. (E) Statistical analysis of invaded or apoptotic GES-1 cells infected with siRNA of REG1A. (F) CCK8 assay analysis of GES-1 cells infected with siRNA of REG1A at 0, 12, 24, 48, and 72 h. * P<0.05, ** P<0.01.

A B MGC-803 BGC-823

** ** ** ** ** ** 40 ** 20 ** ehicle) ehicle) ession ession ** 30 15

eatment/v 20 eatment/v 10 e mRNA expr e mRNA expr SA tr SA tr T T

elativ 10 elativ 5 tio of r tio of r el ( DA C or el ( DA C or

Ra 0 Ra 0 lev Vehicle DAC DAC TSA TSA DAC 5μM+ DAC 5μM+ lev Vehicle DAC DAC TSA TSA DAC 5μM+ DAC 5μM+ 5μM 10μM 300 nM 600 nM TSA TSA 5μM 10μM 300 nM 600 nM TSA TSA 300 nM 600 nM 300 nM 600 nM C D SGC-7901 HGC-27

20 ** 15 * ehicle) ehicle) ession * ession ** 15 10

eatment/v 10 eatment/v e mRNA expr e mRNA expr SA tr SA tr T T 5 elativ 5 elativ tio of r tio of r el ( DA C or el ( DA C or

Ra 0 Ra 0 lev Vehicle DAC DAC TSA TSA DAC 5μM+ DAC 5μM+ lev Vehicle DAC DAC TSA TSA DAC 5μM+ DAC 5μM+ 5μM 10μM 300 nM 600 nM TSA TSA 5μM 10μM 300 nM 600 nM TSA TSA 300 nM 600 nM 300 nM 600 nM

E Vehicle Lenti-REG1A F Vehicle Lenti-REG1A p-Akt p-GSK3β

t-Akt t-GSK3β

GAPDH GAPDH ** 1.2 ** y 1.2 y

0.8 0.8

0.4 0.4 Akt/t-Akt densitometr p- GSH3β/t-GSK3β densitometr

0 p- 0 Vehicle Lenti-REG1A Vehicle Lenti-REG1A

Figure 5. Epigenetic methylation suppresses REG1A expression in GC, and overexpression of REG1A suppresses Akt and GSK3b phosphorylation. (A–D) Relative mRNA expression levels of REG1A in MGC-803 (A), BGC-823 (B), SGC-7901 (C), and HGC-27 (D) cells after treatment with DAC and TSA. (E) Western blotting analysis of phospho-Akt and total-Akt in REG1A overexpressed and control MGC-803 cells. Statistical analysis of phospho-Akt/total-Akt densitometry is shown below. (F) Western blotting analysis of phospho-GSK3b and total-GSK3b in REG1A overexpressed and control MGC-803 cells. Statistical analysis of phospho-GSK3b/total-GSK3b densitometry is shown below. ** P<0.01.

environmental input. However, increasing evidence indicates and increasing apoptosis. Our results show that the PI3K/Akt- that epigenetic changes are responsible for initiation, prolif- GSK3b signaling pathway participates in the regulatory effects eration, invasion, and metastasis of tumor cells by affecting of REG1A on GC cells. 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 Conclusions REG1A in GC. Additionally, we conclude that REG1A was epi- genetically silenced by the aberrant hypermethylation of CpG We found that REG1A overexpression suppressed GC cells in- islands in promoter in GC. The epigenetic regulation needs fur- vasion, viability, and promoted apoptosis. Epigenetic methyl- ther in-depth study. ation suppressed the expression level of REG1A in GC. REG1A exerts its functional roles in GC mainly through the PI3K/Akt PI3K/Akt is a very important signaling pathway involved in and GSK3b signaling pathways. REG1A may be used as a po- various cancers, including GC. GSK3b is a multi-substrate tar- tential therapeutic strategy for GC in future. get protein kinase and also an important downstream regu- lator of the PI3K/Akt pathway. The overexpression of REG1A Conflicts of interest is correlated with decreased p-AKT and p-GSK3b expression, resulting in the suppression of GC cells invasion and viability None.

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