Soft-shelled turtle peptide modulates microRNA profile in human gastric cancer AGS cells

Abstract

Cancer prevention using natural micronutrition on epigenetic mechanisms primarily revolves around plant extracts. However, the role of macronutrition, including animal peptides, on epigenetic modification in cancer has been elusive. In traditional Chinese medicine, the soft-shelled turtle has a long-history of being a functional food that strengthens immunity through unknown mechanisms. The present study aimed to investigate the impact of soft-shelled turtle peptide on microRNA (miRNA) expression in gastric cancer (GC) cells and to analyze the potential anticancer mechanisms for GC. Affymetrix GeneChip miRNA 3.0 Array and quantitative polymerase chain reaction were used to detect the miRNA expression profile in human GC AGS cells treated with the soft-shelled turtle peptide. The results demonstrated that 101 miRNAs (49 upregulated miRNAs and 52 downregulated miRNAs) were significantly differentially expressed in the AGS cells following soft-shelled turtle peptide treatment. Several tumor suppressor miRNAs were upregulated markedly, including miRNA-375, let-7d, miRNA-429, miRNA-148a/148b and miRNA-34a. Pathway analysis indicated that soft-shelled turtle peptide may function with anticancer properties through the Hippo signaling pathway and the forkhead box O signaling pathway. Therefore, these results demonstrated that soft-shelled turtle peptide has the capacity to influence cancer-related pathways through the regulation of miRNA expression in GC cells.

Keywords: gastric cancer cells; macronutrition; microRNA profiling; nutri-epigenetics; soft-shelled turtle peptide.

Figure 1.

Gene Cluster (version 3.0) and Java TreeView software programs were used to perform the hierarchical cluster analysis of differentially expressed miRNA microarray data in AGS cells treated with soft-shelled turtle peptides. A hierarchical cluster analysis of 101 miRNAs whose expression differed significantly between the soft-shelled turtle peptides-treated group (treatment for 72 h) and the untreated group (fold-change >2 or <0.5; P<0.05). Each row represents a miRNA and each column represents a sample. The miRNA expression levels are illustrated using a color key and histogram. The red and green colors denote high and low expression, respectively. miR/miRNA, microRNA.

Figure 2.

RT-qPCR validation of differentially expressed miRNAs that were screened by the microarray assay. Upregulated and downregulated miRNAs are indicated by bars above and below the dotted line respectively. The RT-qPCR results were representative of three independent experiments. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; miRNA, microRNA.

Figure 3.

GO analysis for target genes of differentially expressed miRNAs. To investigate the biological functions of the differentially expressed miRNAs, public online databases were used to obtain miRNA-regulated putative target genes. A GO function analysis of miRNA targets was performed to obtain the significantly over-represented GO terms (P<0.01). GO category for target genes indicated that several important GO terms were possibly associated with cell activation, including intracellular signal transduction, protein phosphorylation, metabolic process and the positive regulation of genes. GO, Gene Ontology; miRNA, microRNA.

Figure 4.

Pathway analysis for target genes of differentially expressed miRNAs. The target genes of soft-shelled turtle peptides related miRNAs were predicted with Kyoto Encyclopedia of Genes and Genomes pathway analysis. miRNA, microRNA; FoxO, forkhead box O; TGF, transforming growth factor; HIF, hypoxia-inducible factor; AMPK, AMP-activated protein kinase; mTOR, mechanistic target of rapamycin.