OTX2 expression contributes progression of gastric cancer in young adults

Abstract

Gastric cancer of young adults is poorly differentiated and has a poor prognosis. However, there are few reports regarding the genetic alteration in gastric cancer of young adults. Bioinformatics methods were used to screen the key genes and signaling pathways of gastric cancer in young adults, and molecular biology techniques were used to verify the key proteins involved in the occurrence and development of gastric cancer in young adults. RNA expression profile microarray data of gastric cancer patients ≤ 45 years old and > 45 years old were downloaded from the TCGA database, and differentially expressed genes were screened by Limma package. GO analysis and KEGG enrichment analysis of DEG were performed in the Gene Function annotation database (DAVID). CytoHubba is used to construct protein interaction networks (PPI) and perform visual analysis to screen out core genes. We obtained 10 hub molecules, including FBXO44, FBXO6, HERC5, FBXL13, FBXO41, NT5E, BMP4, TRIM36, ACAN, ALPL by PPI network with MCODE. Ingenuity Pathway Analysis predicts TBX1, DFNB31, TGFBR3, FBXO44, SLC7A8, DNM1, KITLG, MSH5, MLLT3, DUSP5, ADAMTSL4, ACPP and TBX1 as the transcription factors directly regulated by OTX2. OTX2 had the highest positive expression rate in gastric cancer of young adults by immunohistochemistry. Interference with OTX2 expression inhibits proliferation, migration, invasion and promotes differentiation, apoptosis of NUGC-4 cells (from 35 year old female). Moreover, after interfering OTX2 expression, the downstream proteins and signaling Pathways of OTX2 in NUGC-4 were further analyzed by Transcriptome sequencing and Ingenuity Pathways Analysis. We found interference with OTX2 expression inhibits CEBPB expression and activates calcitriol by IPA analysis, thereby promoting differentiation of NUGC-4. Therefore, OTX2 plays important roles in restraining the differentiation and promoting progression of gastric cancer cells in young adults. Moreover, OTX2/CEBPB signal axis is likely to be a key molecular event in regulating the differentiation of gastric cancer cells in young adults.

Keywords: Differentiation; Gastric cancer; OTX2; Progression; Young adults.

Fig. 1

(A) The volcano plot identified in the gene expression profiling of the young patient group compared with the older group; (B) The significant biological process of these differentially expressed genes involved in regulation of cell morphogenesis and extracellular structure; (C) The top 15 functionally enriched pathways were analysis by KEGG; (D) GO Chord plot displays the relationship between a list of pathway and corresponding genes; (E) Ten hub molecules were found by MCODE analysis; (F) Using Cytoscape, we also found 10 hub molecules; (G) Ingenuity Pathway Analysis predicts TBX1, DFNB31, TGFBR3, FBXO44, SLC7A8, DNM1, KITLG, MSH5, MLLT3, DUSP5, ADAMTSL4, ACPP and TBX1 as the transcription factors directly regulated by OTX2; (H) Using sequence logo to predicted the OTX2 binding sites with DBD.

Fig. 2

(A–B) Signet-ring cell carcinoma is common in gastric cancer of young adults; (C–D, E–F) OTX2 expression was present in the cytoplasm and nucleus of gastric cancer cells of young adults; (G–H) NT5E expression was present in the cytoplasm of gastric cancer cells; (I–J) CXCL17expression was present in the cytoplasm of gastric cancer cells; (K–L) FBXO44 was present in the cytoplasm of gastric cancer cells. (M–N, O–P) OTX2 expression was negative in the normal gastric tissues of young adults.

Fig. 3

The efects of OTX2 expressionon on biologic processes in NUGC-4 cells after downregulated OTX2 expression. (A–C) The morphology of some NUGC-4 cells changed from round to spindle cells after interference with OTX2 expression; (D–F, T) Interference of NUGC-4 expression attenuated colony formation in NUGC-4 cells. (*P < 0.05); (S) Compared with SiRNA1 and siRNA3, siRNA2 had the best effect on interfering OTX2 expression. The upper bands represent OTX2 expression levels. The bands below represent β-actin expression levels. (G–I, V) Knocking-down expression of OTX2 increased the rate of apoptosis of NUGC-4 cells (*P < 0.05); (J–L, U) Compared with cells transfected with control siRNA (NC-siRNA), interference of OTX2 expression markedly inhibited the invasion of NUGC-4 cells; (M–R) Interference of OTX2 expression by siRNA inhibited migration of NUGC-4 cells.

Fig. 4

(A) Disease Ontology enrichment analysis showed that the signaling pathways associated with germ cell cancer and embryonal cancer were correlated with the down-regulation of OTX2 expression; (B) KEGG enrichment analysis showed that signaling pathways related to DNA replication and cell cycle were associated with down-regulation of OTX2 expression; (C) Cell cycle Control of Chromosomal Replication was significantly inhibited after inhibition of OTX2 expression; (D) IPA showed that CEBPB was significantly inhibited; (E) CEBPB was predicted to be strongly repressed, and there were 52 consistently repressed genes after inhibition of OTX2 expression; (F) CEBPB expression was decreased in NUGC-4/siRNA-OTX2 cells.