PHTF2 regulates lipids metabolism in gastric cancer

Abstract

Identification of hub genes and key pathways of gastric cancer was recognized to be essential to elucidate the tumorigenesis of GC. This study was aimed to identify the differentially expressed genes (DEGs) in GC via bioinformatics methods and their related pathways involved in the pathological process of GC. Gene expression profile datasets acquired by microarray chips or RNA-seq were downloaded from GEO dataset and TCGA, and 298 differentially expressed genes was identified. The Gene Ontology (GO) and Kyoto Gene and Genomic Encyclopedia (KEGG) pathways of DEGs were then analyzed by the DAVID database to elucidate the potential molecular functions of DEGs. The protein-protein interaction (PPI) network of DEGs was further analyzed with the STRING database and PHTF2 was identified as a hub gene in the PPI network. Subsequently, PHTF2 was found to be highly expressed in different subtypes of gastric cancer tissues obtained from TCGA database or clinical patients, resulting with a poor prognosis. By GSEA, PHTF2 was found to significantly enrich the fatty acid metabolism pathway in gastric cancer. Moreover, PHTF2-regulated lipids metabolism significantly affected the tumorigenesis of GC cells. In summary, this work identified a new mechanism by which PHTF2 precipitated in the pathological process of GC by regulating cellular lipid metabolism.

Keywords: PHTF2; gastric cancer; lipids metabolism; tumorigenesis.

Figure 1

PHTF2 was identified as a hub gene in GEO datasets and TCGA database. (A) Venn chart of the different-expressing genes in GSE79973, GSE19826 and TCGA gastric cancer datasets, which overlapped according to the analysis of FunRich software. (B) Pie chart for illustration of different-expressing genes molecular pathways and process analyzed by DAVID online tools. (C, D) Molecular function and KEGG pathway enrichment of differentially expressed genes analyzed by DAVID online tools. (E) On line tool of STRING database analysis of the PPI network for the different-expressing genes. (F) Amplification of the network for PPI associated with PHTF2.

Figure 2

PHTF2 was significantly up-regulated in gastric cancer. (A) Expression pattern of PHTF2 in differential human malignancies from “Oncomine” database. (B) PHTF2 was significantly up-regulated in different histological subtypes of gastric cancer. (C) PHTF2 mRNA expressed in four different stages of cancerous tissues compared to para-cancerous tissues based on TCGA gastric cancer dataset. (D) Overall survival of patients with gastric cancer was calculated using Kaplan–Meier analysis according to the PHTF2 mRNA expression lever. (E) PHTF2 is commonly up-regulated in various gastric cancer cell lines from CCLE database. (F) PHTF2 mRNA expression pattern in 50 pairs of human gastric cancer tissues (Cancer) and adjacent tissues (Normal). (G) The expression pattern of ACSL1 and PHTF2 was associated in gastric cancer tissues. (H) Representative IHC image show PHTF2 protein expression pattern in gastric cancer tissues. (I) Different distribution of PHTF2 was statistically analyzed with Chi-square test.

Figure 3

Effects of PHTF2 on the intracellular contents of lipids and key lipid metabolic enzymes in gastric cancer cells. (A) Identification of gene sets enriched in phenotypes correlated with PHTF2 by GSEA using TCGA data. (B) Heat map of core enrichment genes in the gene set KEGG FATTY ACID METABOLISM. (C) Up: Western blot showed PHTF2 protein expression in indicated cells. Down: RT-qPCR analysis for mRNA levels of the key lipid metabolic enzymes ACSL2, ACSL3, HADH, ADH4 and ALDH1B in the indicated cells. (D, E) Cellular content of triglycerides (D) and phospholipids (E) was detected in the indicated cells. (F) The neutral lipids content was detected by double staining with BODIPY 493/503 dye in the indicated cells. Scale bars (10 μm). (G, H) Cellular content of triglycerides (G) and phospholipids (H) was detected in the indicated cells. (I) The neutral lipids content was detected by double staining with BODIPY 493/503 dye in the indicated cells. Scale bars (10 μm). (J) RT-qPCR analysis for ACSL1 mRNA levels in the indicated cells.

Figure 4

PHTF2-regulated fatty acid metabolism promotes tumorigenic ability of gastric cancer cells in vitro and in vivo. (A) CCK8 assay showed cells viability in the indicated cells. (B) Colony formation assay showed cell growth of the indicated cells. (C) Qualification of the colony formation shown in B (n=3). (D) The representative pictures of dissected tumors from nude mice transplanted with indicated cells. (E) Subcutaneous tumor growth curves of mice in different treatment groups was presented. (F) The average weight of tumors at the time the animals were sacrificed in the indicated groups. (G) Relative mRNA expression pattern of associated gene in tumor tissue acquired from nude mice.