Esomeprazole affects the proliferation, metastasis, apoptosis and chemosensitivity of gastric cancer cells by regulating lncRNA/circRNA-miRNA-mRNA ceRNA networks

Abstract

Recently, proton pump inhibitors have become a hot research topic in the field of cancer drug research. However, the specific anti-tumor effect and underlying mechanisms of esomeprazole (ESO) in gastric cancer (GC) have remained elusive. In the present study, the toxic effects of ESO on the GC cell line AGS were investigated. MTT assays confirmed that ESO inhibited the proliferation of AGS cells and significantly enhanced their chemosensitivity. Transwell assays were performed to determine the anti-metastatic effects of ESO in AGS cells. Flow cytometry demonstrated that ESO induced cell apoptosis and caused cell cycle arrest in the S and G2/M phases. Furthermore, the differential expression of 948 long non-coding RNAs (lncRNAs), 114 circular RNAs (circRNAs), 1,197 mRNAs and 199 microRNAs (miRNAs) was detected in AGS cells via microarray analysis and RNA-sequencing. The top 10 differently expressed genes were mostly located on chromosomes 10 and 19. In addition, Gene Ontology analysis indicated that the genes were accumulated in functional terms associated with DNA replication, the cell cycle and the apoptotic signaling pathway. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed a variety of significantly dysregulated signaling pathways and targets, including the EGFR tyrosine kinase inhibitor resistance pathway, forkhead box O signaling pathway, p53 signaling pathway and platinum drug resistance pathway. Subsequently, the interactions of microtubule-associated protein 2 (MAP2), homeodomain-interacting protein kinase 2 (HIPK2) and ankyrin 2 (ANK2) were noted in a competing endogenous RNA (ceRNA) network, which may be important targets of ESO, exerting an anti-tumor effect in AGS cells. Collectively, ESO affects the proliferation, metastasis, apoptosis and chemosensitivity of gastric cancer cells by regulating long non-coding RNA/circRNA-miRNA-mRNA ceRNA networks.

Keywords: apoptosis; chemosensitivity; competing endogenous RNA; esomeprazole; gastric cancer; metastasis; proliferation.

Figure 1.

ESO inhibits proliferation and metastasis, and increases chemosensitivity in AGS cells. (A-C) MTT assays demonstrated that (A) ADM, (B) DDP and (C) ESO inhibited cell proliferation. (D) The IC₅₀ of ADM, DDP and ESO in AGS cells. (E and F) ESO significantly increased chemosensitivity in AGS cells in a dose- and time-dependent manner (E) 24 h and (F) 48 h. (G and H) Transwell assays verified that ESO inhibited the (G) migration and (H) invasion of AGS cells, alone or in combination with 0.2 µg/ml ADM or 20 µg/ml DDP (scale bars, 50 µm). *P<0.05, **P<0.01 and ***P<0.001 vs. control; ^###P<0.001 as indicated. ESO, esomeprazole; ADM, adriamycin; DDP, cisplatin; IC₅₀, half maximal inhibitory concentration.

Figure 2.

ESO induces AGS cell apoptosis by causing cell cycle arrest at the S and G2/M phases. (A) ESO induced AGS cell apoptosis, as analyzed by Annexin V-FITC/PI staining. (B) ESO caused cell cycle arrest at the S and G2/M phases in a time-dependent manner. (C-E) ESO combined with ADM and DDP induced S-phase arrest in AGS cells after incubation for (C) 12, (D) 24 and (E) 48 h. *P<0.05, **P<0.01 and ***P<0.001 vs. control. Ns, no significance; ESO, esomeprazole; ADM, adriamycin; DDP, cisplatin.

Figure 3.

Differentially expressed genes and transcripts in AGS cells treated with ESO. (A) Statistical analysis of the results of differentially expressed genes and transcripts. (B) Chromosome distribution of the top 10 differentially expressed lncRNAs, circRNAs and mRNAs. Hierarchical clustering analysis of (C) lncRNAs, (D) circRNAs, (E) mRNAs and (F) miRNAs. Groups: A, control group; B, experimental group (40 µg/ml ESO). ESO, esomeprazole; chr, chromosome; lncRNA, long non-coding RNA; circRNA, circular RNA; miRNA, microRNA.

Figure 4.

Enrichment analysis of differentially expressed microRNAs in the categories biological process, cellular component and molecular function.

Figure 5.

Top 20 most significantly enriched signaling pathways of differentially expressed microRNAs. Each bubble corresponds to a pathway. The bubble scale represents the number of differentially expressed genes. The color of the bubble represents the P-value. GnRH, gonadotropin-releasing hormone; ECM, extracellular matrix; cGMP, cyclic guanosine monophosphate; PKG, protein kinase G; MAPK, mitogen-activated protein kinases.

Figure 6.

Functional enrichment analysis of components of the lncRNA/circRNA-miRNA-mRNA ceRNA network. (A and B) Enrichment analysis of the (A) lncRNA-miRNA-mRNA ceRNA network and the (B) circRNA-miRNA-mRNA ceRNA network in the GO terms biological process, cellular component and molecular function. (C and D) Top 10 most significantly enriched signaling pathways of the (C) lncRNA-miRNA-mRNA ceRNA and (D) circRNA-miRNA-mRNA ceRNA networks. lncRNA, long non-coding RNA; circRNA, circular RNA; miRNA, microRNA; ceRNA, competing endogenous RNA; GO, gene ontology; HTLV1, human T-cell leukemia virus type 1; rDNA, ribosomal DNA; hsa, Homo sapiens.

Figure 7.

lncRNA/circRNA-miRNA-mRNA ceRNA network. (A) lncRNA-miRNA-mRNA ceRNA network. (B) circRNA-miRNA-mRNA ceRNA network. Green represents lncRNAs; yellow represents circRNAs; blue represents miRNAs; red represents mRNAs. ceRNA, competing endogenous RNA; lncRNA, long non-coding RNA; circRNA, circular RNA; miRNA, microRNA; hsa, Homo sapiens; ENST, the symbol of Ensembl ID.