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 clin- icopathological findings or patient prognoses. REG1A overexpression could suppress the invasion, cell viabil- ity 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 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) 5834 [Chemical Abstracts/CAS] [Index Copernicus] 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 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 stain- ing, 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 significant- ly 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 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) 5835 [Chemical Abstracts/CAS] [Index Copernicus] Qiu Y.-S. et al.: LAB/IN VITRO RESEARCH DNA methylation-mediated silencing of REG1A affects gastric cancer prognosis © Med Sci Monit, 2017; 23: 5834-5843 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’-deoxycyti- dine (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.