FBXO32 promotes gastric cancer progression by regulating NME1

Abstract

Background: Gastric cancer, a common cancer, has a high incidence rate, the 5-year survival rate of gastric cancer with distant metastasis is less than 10%, so finding new targets for gastric cancer is of clinical importance. As a member of the F-box protein family, F-box protein 32 (FBXO32) plays a role in various cancers, but its role in gastric cancer is currently unclear, this study mainly focused on the role and mechanism of FBXO32 in gastric cancer.

Methods: The FBXO32 expression was found through The Cancer Genome Atlas (TCGA) database and the Gene Expression Omnibus (GEO) database. The FBXO32 expression in cell lines was found through Western blot assay. Cell cloning assay and Cell Counting Kit-8 (CCK8) assay were used to investigate the effect of FBXO32 on gastric cancer cell proliferation. Wound-healing assay and transwell assay were used to research the effect of FBXO32 on gastric cancer cell metastasis. Cancer stem cell sphere-forming assay was used to find out the effect of FBXO32 on the stemness of gastric cancer cells. Nude mouse tumorigenesis assay was used to investigate the effect of FBXO32 on gastric cancer tumor growth.

Results: It was found that FBXO32 expression is elevated in gastric cancer, which is associated with poor prognosis. In addition, FBXO32 expression is also elevated in gastric cancer cell lines. The knockdown of FBXO32 can inhibit the proliferation, migration, invasion and stemness of gastric cancer cells, inhibit the subcutaneous tumor formation of gastric cancer cells in nude mice, and increase the expression of non-metastatic cells 1 (NME1). Low expression of NME1 is associated with poor prognosis in gastric cancer. The knockdown of NME1 expression can offset part of the tumor suppressor activity of the knockdown of FBXO32.

Conclusions: It is believed that FBXO32 promotes the progression of gastric cancer by regulating NME1. Most importantly, our findings could provide information for the mechanism of gastric cancer and the discovery of new targets.

Keywords: F-box protein 32 (FBXO32); gastric cancer; non-metastatic cells 1 (NME1).

Figure 1

High expression of FBXO32 is associated with poor prognosis in gastric cancer patients, FBXO32 expression is elevated in gastric cancer tissues and cell lines. (A) The relationship between FBXO32 expression and prognosis of gastric cancer patients in the Kaplan-Meier Plotter database. (B-D) The relationship between FBXO32 expression and prognosis of gastric cancer patients in the R2 database. (E) Expression of FBXO32 in gastric cancer tissues in GEPIA database. (F) Expression of FBXO32 in gastric cancer tissues in GSE13911. (G) Expression of FBXO32 in gastric cancer tissues in GSE19826. (H) Expression of FBXO32 in gastric cancer tissues in GSE54129. (I) Expression of FBXO32 in gastric cancer tissues in GSE63098. (J) Expression of FBXO32 in gastric cancer tissues in GSE65081. (K) Expression of FBXO32 in gastric cancer tissues in GSE79973. (L) Expression of FBXO32 in gastric cancer tissues in GSE81948. (M) Expression of FBXO32 protein in gastric cancer cell lines and gastric epithelial cells. (N) Quantification of FBXO32 protein expression in gastric cancer cell lines and gastric epithelial cells. *, P<0.05; **, P<0.01. CI, confidence interval; FBXO32, F-box protein 32; GEPIA, Gene Expression Profiling Interactive Analysis; HR, hazard ratio; STAD, stomach adenocarcinoma.

Figure 2

Knockdown efficiency of FBXO32 and reduced expression of FBXO32 inhibits the proliferation of gastric cancer cells. (A) Expression of green fluorescent protein of transfected cells and non-transfected cells. (B) Expression of FBXO32 protein after knockdown. (C) Quantification after FBXO32 protein knockdown. (D) Cell cloning experiments of NC group and FBXO32 knockdown group. 1,000 FBXO32 knockdown group cells and NC group cells were placed in a six-well plate, complete culture medium was added, and crystal violet staining was performed after 10 days to count the number of spheres in each well. (E) Statistics of cell cloning experiments. (F) CCK8 assay of NC group and FBXO32 knockdown group. (G) Expression of CDK2 and CDK6 proteins after FBXO32 protein knockdown. (H) Quantification of CDK2 protein after FBXO32 protein knockdown. (I) Quantification of CDK6 protein after FBXO32 protein knockdown. *, P<0.05; **, P<0.01; ***, P<0.001 (n=3). FBXO32, F-box protein 32; shRNA-NC, short hairpin RNA-negative control.

Figure 3

Knockdown of FBXO32 expression inhibits migration, invasion and stemness of gastric cancer cells. (A) Wound-healing assay of NC group and FBXO32 knockdown group (scale bar =100 μm, direct observation under a ×10 microscope). (B) Statistics of wound-healing assay. (C) Transwell invasion assay of NC group and FBXO32 knockdown group (crystal violet staining, scale bar =100 μm). (D) Statistics of transwell invasion assay. (E) Cancer stem cell sphere-forming assay of NC group and FBXO32 knockdown group (scale bar =100 μm). 1,000 FBXO32 knockdown group cells and NC group HGC27 cells were cultured in low-adhesion six-well plates with serum free DMEM-F12 supplemented with 20 ng/mL EGF and 10 ng/mL FGF2 for 10 days. The number of spheres in each well was observed under a microscope. (F) Statistics of cancer stem cell sphere-forming assay. (G) Expression of MMP2, MMP9, CD44 and LGR5 proteins of NC group and FBXO32 knockdown group. (H) Quantification of expression of MMP2, MMP9, CD44 and LGR5 proteins. *, P<0.05; **, P<0.01; ***, P<0.001 (n=3). FBXO32, F-box protein 32; shRNA-NC, short hairpin RNA-negative control.

Figure 4

Knockdown of FBXO32 inhibits gastric cancer tumor growth and increases NME1 expression. (A) Photo of gastric cancer tumor in nude mice of NC group and FBXO32 knockdown group. (B) Volumetric statistics of gastric cancer tumors. (C) Gastric cancer tumor weight statistics. (D) Immunohistochemistry of NME1 protein in gastric cancer tumors of NC group and FBXO32 knockdown group (DAB staining, scale bar =20 μm). (E) Quantification of NME1 protein by immunohistochemistry in gastric cancer tumors. (F) Western blot assay of NME1 protein in gastric cancer cell lines. (G) Quantification of NME1 protein in gastric cancer cell lines HGC27 by Western blot assay. (H) Quantification of NME1 protein in gastric cancer cell lines AGS by Western blot assay. *, P<0.05; **, P<0.01. DAB, diaminobenzidine; FBXO32, F-box protein 32; NME1, non-metastatic cells 1; shRNA-NC, short hairpin RNA-negative control.

Figure 5

Knockdown of NME1 expression can offset part of the tumor suppressor activity of knockdown of FBXO32. (A) The relationship between NME1 expression and prognosis of gastric cancer patients in the Kaplan-Meier plotter database. (B) The relationship between NME1 expression and prognosis of gastric cancer patients in the R2 database. (C) Protein expression after NME1 knockdown. (D) Quantification of protein expression after NME1 knockdown. (E) Wound-healing assay (scale bar =100 μm, direct observation under a ×10 microscope). (F) Statistics of wound-healing. (G) Statistics of transwell invasion assay. (H) Statistics of cancer stem cell sphere-forming assay. (I) Statistics of cell cloning experiments. (J) Transwell invasion assay (crystal violet staining, scale bar =100 μm). (K) Cancer stem cell sphere-forming assay (scale bar =100 μm). 1,000 HGC27 cells were cultured in low-adhesion six-well plates with serum free DMEM-F12 supplemented with 20 ng/mL EGF and 10 ng/mL FGF2 for 10 days. The number of spheres in each well was observed under a microscope. (L) Cell cloning experiments. 1,000 HGC27 cells were placed in a six-well plate, complete culture medium was added, and crystal violet staining was performed after 10 days to count the number of spheres in each well. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001 (n=3). CI, confidence interval; FBXO32, F-box protein 32; NME1, non-metastatic cells 1; shRNA-NC, short hairpin RNA-negative control; siRNA-NC, small interfering RNA-negative control.