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

Abstract

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 AND 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.

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.

Figure 2

Overexpression of REG1A reduces the cell viability of GC cells. (A) The mRNA expression level of REG1A in 6 GC cell lines and human gastric epithelial cell line GES-1. (B, C) REG1A mRNA expression level in MGC-803 (B) and BGC-823 (C) GC cells, which were infected with lenti-vector or lenti-REG1A. (D, E) REG1A protein expression level in MGC-803 (D) and BGC-823 (E) GC cells, which were infected with lenti-vector or lenti-REG1A. Statistical analysis of REG1A expression in the 2 groups is shown below. (F) CCK8 assay analysis of MGC-803 cells infected with lenti-vector or lenti-REG1A at 0, 12, 24, 48, and 72 h. (G) CCK8 assay analysis of BGC-823 cells infected with lenti-vector or lenti-REG1A at 0, 12, 24, 48, and 72 h. ** P<0.01.

Figure 3

REG1A overexpression suppresses the invasion and supports the apoptosis of GC cells. (A) Representative photos of invaded MGC-803 cells infected with lenti-vector or lenti-REG1A. Statistical analysis of invaded MGC-803 cells in the 2 groups is shown right. Scale bars: 10 μm. (B) Representative photos of invaded BGC-823 cells infected with lenti-vector or lenti-REG1A. Statistical analysis of invaded BGC-823 cells in the 2 groups is shown right. Scale bars: 10 μm. (C) Flow cytometry analysis of apoptotic MGC-803 cells infected with lenti-vector or lenti-REG1A. Statistical analysis of apoptotic MGC-803 cells in the 2 groups is shown right. (D) Flow cytometry analysis of apoptotic BGC-823 cells infected with lenti-vector or lenti-REG1A. Statistical analysis of apoptotic BGC-823 cells in the 2 groups is shown at right. ** P<0.01.

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.

Figure 5

Epigenetic methylation suppresses REG1A expression in GC, and overexpression of REG1A suppresses Akt and GSK3β 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-GSK3β and total-GSK3β in REG1A overexpressed and control MGC-803 cells. Statistical analysis of phospho-GSK3β/total-GSK3β densitometry is shown below. ** P<0.01.