FBXO28 promotes cell proliferation, migration and invasion via upregulation of the TGF-beta1/SMAD2/3 signaling pathway in ovarian cancer

Abstract

Background: Ovarian cancer is one of the most common gynecological malignancies due to the lack of early symptoms, early diagnosis and limited screening. Therefore, it is necessary to understand the molecular mechanism underlying the occurrence and progression of ovarian cancer and to identify a basic biomarker for the early diagnosis and clinical treatment of ovarian cancer.

Methods: The association between FBXO28 and ovarian cancer prognosis was analyzed using Kaplan‒Meier survival analysis. The difference in FBXO28 mRNA expression between normal ovarian tissues and ovarian tumor tissues was obtained from The Cancer Genome Atlas (TCGA), and Genotype-Tissue Expression (GTEx) cohorts. The expression levels of the FBXO28 protein in ovarian cancer tissues and normal ovarian tissues were measured via immunohistochemical staining. Western blotting was used to determine the level of FBXO28 expression in ovarian cancer cells. The CCK-8, the colony formation, Transwell migration and invasion assays were performed to evaluate cell proliferation and motility.

Results: We found that a higher expression level of FBXO28 was associated with poor prognosis in ovarian cancer patients. Analysis of the TCGA and GTEx cohorts showed that the FBXO28 mRNA level was lower in normal ovarian tissue samples than in ovarian cancer tissue samples. Compared with that in normal ovarian tissues or cell lines, the expression of FBXO28 was greater in ovarian tumor tissues or tumor cells. The upregulation of FBXO28 promoted the viability, proliferation, migration and invasion of ovarian cancer cells. Finally, we demonstrated that FBXO28 activated the TGF-beta1/Smad2/3 signaling pathway in ovarian cancer.

Conclusions: In conclusion, FBXO28 enhanced oncogenic function via upregulation of the TGF-beta1/Smad2/3 signaling pathway in ovarian cancer.

Keywords: FBXO28; Migration; Ovarian cancer; Smad2/3; TGF-b1.

Fig. 1

FBXO28 expression is correlated with ovarian cancer prognosis. A. TCGA combined with GTEx analysis showed higher expression levels of FBXO28 in ovarian cancer tissues than in normal ovarian tissues. B. Representative images of immunohistochemical staining for FBXO28 in ovarian cancer and normal ovarian tissue. Scale bar: 100 μm. C. IHC staining results for FBXO28 in ovarian cancer tissues and normal ovarian samples. D. Expression level of FBXO28 in normal ovarian cells and ovarian cancer cells determined by Western blotting

Fig. 2

FBXO28 overexpression promotes the viability of ovarian cancer cells. A. Left panel: The efficacy of FBXO28 knockdown was determined using Western blotting. Right panel: Corresponding quantitative analysis results. B. Left panel: The efficacy of FBXO28 overexpression was determined using Western blotting. Right panel: Corresponding quantitative analysis results. C. A CCK-8 assay showed that FBXO28 knockdown suppressed the viability of ovarian cancer cells. D. A CCK-8 assay showed that FBXO28 overexpression promoted the viability of ovarian cancer cells. E. Left panel: FBXO28 knockdown decreased the proliferation capacity of A2780 cells, as determined by a colony formation assay. Right panel: Corresponding quantitative analysis results. F. Left panel: FBXO28 overexpression increased the proliferation capacity of A2780 cells, as determined by a colony formation assay. Right panel: Corresponding quantitative analysis results. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 3

FBXO28 promotes the migration and invasion of ovarian cancer cells. A. Transwell migration and invasion assays showed that knockdown of FBXO28 after 36 h inhibited the migration and invasion of ovarian cancer cells. B. The quantitative analysis results obtained from Figure A. C. Transwell migration and invasion assays showed that overexpression of FBXO28 after 24 h promoted the migration and invasion of ovarian cancer cells. D. The quantitative analysis results obtained from Figure C. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 4

FBXO28 regulates TGF-b1/Smad2/3 pathway in ovarian cancer cells. A. KEGG enrichment analysis showed that FBXO28 may act on ovarian cancer via the TGF-b1 signaling pathway. B. Western blot analysis of TGF-b1 pathway expression after FBXO28 was knocked down in A2780 cells. C. Western blot analysis of TGF-b1 pathway expression after FBXO28 was knocked down in SKOV3 cells. D. The quantitative analysis results obtained in Figure B and C. E. Western blot analysis of TGF-b1 pathway expression after overexpression of the FBXO28 gene in ovarian cancer cells. F-G. The quantitative analysis results for Figure E. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

FBXO28 regulates the expression of EMT markers. A. Western blot analysis of EMT marker expression after FBXO28 was knocked down in ovarian cancer cells. B. The quantitative analysis results obtained in Figure A. C. Western blotting analysis of EMT marker expression after FBXO28 overexpression in ovarian cancer cells. D. The quantitative analysis results obtained in Figure C. *P < 0.05, ** P < 0.01

Fig. 6

TGF-b1 overexpression rescues FBXO28 knockdown-mediated suppression of cell viability. A. The expression of TGF-b1, Smad2/3 and p-Smad2/3 in ovarian cancer cells was measured by western blotting after FBXO28 knockdown and TGF-b1 overexpression. B. The quantitative analysis results obtained in Figure A. C. A CCK-8 assay showed that overexpression of the TGF-b1 gene reversed the FBXO28 knockdown-mediated decrease in ovarian cancer cell viability. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 7

TGF-b1 overexpression rescues FBXO28 knockdown-mediated suppression of migration and invasion. A. Transwell migration and invasion assays were performed in ovarian cancer cells after FBXO28 knockdown and TGF-b1 overexpression. B. The quantitative analysis results obtained in Figure A. *P < 0.05, ** P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 8

FBXO28 knockdown inhibits tumor growth in vivo. A. Images of xenograft tumors obtained 34 days after injection of SKOV3-knockdown cells or corresponding control cells in nude mice. B. Changes in tumor volume measured at 4-day intervals after injection of SKOV3-knockdown cells or corresponding control cells in nude mice. Tumor volume = (L×W2)/2. CC. Images of the collected graft tumors. D. The weight of each xenograft tumor. E. Representative images of subcutaneous tumor samples from the SKOV3 knockdown and corresponding control groups subjected to immunohistochemical staining for the FBXO28, N-cadherin, E-cadherin and Ki67 proteins. **P < 0.01. F: Left panel: Relationship between FBXO28 expression and PFS in ovarian cancer patients. Right panel: correlation between FBXO28 expression and OS in ovarian cancer patients. **P < 0.01.