FHOD1 is upregulated in gastric cancer and promotes the proliferation and invasion of gastric cancer cells

Abstract

Gastric cancer (GC) is one of the main causes of cancer-associated morbidity and mortality worldwide. The present study aimed to investigate the role of the gene encoding formin homology 2 domain containing 1 (FHOD1) protein in GC development. Data from The Cancer Genome Atlas were firstly analyzed, and immunohistochemistry was conducted on GC tissues. The results demonstrated that FHOD1 expression in GC tissues was significantly increased compared with adjacent non-tumor tissues. Furthermore, the expression level of FHOD1 was negatively associated with the overall survival of patients with GC. For the functional studies, lentivirus-mediated short hairpin RNA against FHOD1 and FHOD1-overexpression vectors were constructed to knockdown and overexpress the expression level of FHOD1 in human GC cell lines, respectively. The results indicated that FHOD1 knockdown inhibited the proliferation, colony formation and migratory and invasive abilities of GC cells. Conversely, overexpression of FHOD1 in GC cells promoted soft-agar colony formation and migratory and invasive abilities. In addition, it was demonstrated that genes of which expression levels were correlated with FHOD1 were enriched in the Gene Ontology term of 'extracellular matrix (ECM) structural constituent', suggesting that FHOD1 may serve an important role in the regulation of ECM. In conclusion, the present study demonstrated that FHOD1 may exert an oncogenic role in cultured GC cells and be inversely associated with the overall survival of patients with GC.

Keywords: FHOD1; gastric cancer; invasion; overall survival; overexpression; proliferation; short hairpin RNA.

Figure 1.

FHOD1 is upregulated in tumor tissues compared with non-tumor tissues in GC. (A) Expression level of FHOD1 in GC tissues compared with adjacent non-tumor tissues in The Cancer Genome Atlas dataset. (B) Log2FC of the expression level of FHOD1 in GC tissues compared with adjacent non-tumor tissues. (C) Expression level of FHOD1 in GC tissues compared with adjacent non-tumor tissues based on immunohistochemistry (magnification, ×200). GC, gastric cancer; FHOD1, formin homology 2 domain containing 1.

Figure 2.

Prognostic value of FHOD1 expression for patients with GC was assessed using Kaplan-Meier analysis. Original expression level data were from our previous panomics-based experiments (18). GC, gastric cancer; FHOD1, formin homology 2 domain containing 1; HR, hazard ratio.

Figure 3.

FHOD1 expression levels in HGC27 cells after shFHOD1 or LV-FHOD1 virus transfection, and in MKN45 cells after LV-FHOD1 virus transfection. (A) FHOD1 mRNA and protein expression levels in HGC27 cells were detected via RT-qPCR (left) and western blotting (right). (B) FHOD1 mRNA and protein expression levels in MKN45 cells were detected via RT-qPCR (left) and western blotting (right). ***P<0.001. FHOD1, formin homology 2 domain containing 1; RT-qPCR, reverse transcription-quantitative PCR; sh, short hairpin RNA. Control, untransfected cells; NC, negative control; LV, lentivirus.

Figure 4.

Proliferation of gastric cancer cells after knockdown and overexpression of FHOD1. Cell Counting Kit-8 cell proliferation assay demonstrated that the proliferative rate was significantly decreased at each time point after FHOD1 knockdown in HGC-27 cells. ***P<0.001. FHOD1, formin homology 2 domain containing 1; sh, short hairpin; OD, optical density; LV, lentivirus.

Figure 5.

Cell cycle distribution measured via flow cytometry in GC cells after FHOD1 knockdown and overexpression. (A) Representative plots for the Cell Cycle Phase detection for each of the 3 groups. (B) Quantification of GC cell cycle distribution measured via flow cytometry in GC cells after knockdown and overexpression of FHOD1. Proportion of cells in S phase after FHOD1 knockdown was significantly decreased and that of cells in G1 phase after FHOD1 overexpression was markedly decreased. **P<0.01 and ***P<0.001. FHOD1, formin homology 2 domain containing 1; sh, short hairpin; GC, gastric cancer; LV, lentivirus.

Figure 6.

Efficiency of HGC-27 colony formation following shFHOD1 or LV-FHOD1 transfection for 2 weeks. (A) Cell colony growth of HGC-27 cells in petri dishes in the control, shFHOD1 and LV-FHOD1 groups. (B) Quantification of cell colony numbers of HGC-27 cells in the control, shFHOD1 and LV-FHOD1 groups. ***P<0.001. sh, short hairpin RNA; FHOD1, formin homology 2 domain containing 1; sh, short hairpin; LV, lentivirus.

Figure 7.

Migratory ability of HGC-27 cells after FHOD1 knockdown and overexpression. (A) Migratory potential of stably transfected HGC27 cells was examined using a cell wound scratch assay. Images were captured at time intervals of 0 and 24 h (magnification, ×40). (B) Percentage of migration was statistically analyzed. *P<0.05 and **P<0.01. FHOD1, formin homology 2 domain containing 1; sh, short hairpin; LV, lentivirus.

Figure 8.

Invasive ability of GC cells after knockdown and overexpression of FHOD1 with or without Matrigel. (A) Left: The number of HGC-27 cells crossing the basement membrane with Matrigel (magnification, ×100). Right: Quantification of GC cells crossing the basement membrane after knockdown and overexpression of FHOD1. (B) Left: The number of HGC-27 cells crossing the basement membrane without Matrigel (magnification, ×100). Right: Quantification of GC cells crossing the basement membrane after knockdown and overexpression of FHOD1. **P<0.01 and ***P<0.001. FHOD1, formin homology 2 domain containing 1; GC, gastric cancer; sh, short hairpin; LV, lentivirus.

Figure 9.

Viability of MKN45 cells after FHOD1 overexpression was analyzed using a soft agar colony formation assay (magnification, ×100). ***P<0.001. FHOD1, formin homology 2 domain containing 1; NC, negative control; LV, lentivirus.

Figure 10.

Functional enrichment analysis of genes positively co-expressed with FHOD1 using WebGestalt (Web-based Gene Set analysis Toolkit). FHOD1, formin homology 2 domain containing 1.

Figure 11.

Expression levels of ECM-related genes and FHOD1 in HGC-27 and MKN-45 spheroids. (A) COL1A1 and COL18A1 expression in HGC-27 spheroids after FHOD1 overexpression. (B) COL1A1 and COL18A1 expression in MKN-45 spheroids after FHOD1 overexpression. ***P<0.001. FHOD1, formin homology 2 domain containing 1; COL1A1, collagen type I alpha 1 chain; COL18A1, collagen type XVIII alpha 1 chain.