OSR1 downregulation indicates an unfavorable prognosis and activates the NF-κB pathway in ovarian cancer

Abstract

Background: Odd-skipped related 1 (OSR1) has been reported as a tumor suppressor gene in various malignant tumors. The mechanism through which OSR1 regulates ovarian cancer (OC) progression remains unclear.

Materials and methods: Immunohistochemistry was utilized to evaluate OSR1 expression in patients with ovarian cancer. We investigated the association between clinicopathological parameters and OSR1 expression in OC patients and the influence of OSR1 expression on patient survival and prognosis. OC cells with OSR1 overexpression or knockdown were established and validated using Western blot and Quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The influence of OSR1 on the NF-κB pathway was examined by analyzing the p-IκBα, IκBα, p65, and p-p65 protein expression. In vitro assays, such as cell cycle assay, Cell Counting Kit-8 (CCK-8), transwell invasion assay, wound healing migration assay, enzyme-linked immunoassay (ELISA), and Annexin V/PI flow cytometry apoptosis assay, were conducted to explore the effect of OSR1 knockdown or dual inhibition of OSR1 and the NF-κB pathway on OC malignant biological behavior.

Results: OSR1 expression was downregulated in OC tissues, with significant associations observed between its expression and The International Federation of Gynecology and Obstetrics (FIGO) stage and tissue differentiation. Low OSR1 expression in OC patients correlated with reduced overall survival (OS) rates and poor prognosis. In vitro, experiments confirmed a negative correlation between OSR1 expression and NF-κB pathway activity. OSR1 knockdown facilitated OC cell malignant biological behavior, while the NF-κB pathway inhibitor (Bay 11-0782) reversed the impacts of OSR1 knockdown on cell proliferation, migration, invasion, and apoptosis.

Conclusion: Our findings indicate that OSR1 is downregulated and associated with OC prognosis. OSR1 suppresses NF-κB pathway activity and inhibits OC progression by targeting the NF-κB pathway.

Keywords: Downregulation; NF-κB pathway; OSR1; Ovarian cancer; Unfavorable prognosis.

Fig. 1

Low expression of OSR1 was shown in ovarian cancer tissues and was correlated with worse OS. A The representive image of OSR1 low expression in OC tissue by IHC (200 × and 400 ×); B The representive image of OSR1 high expression in normal ovarian tissue by IHC (200 × and 400 ×); C Statistical diagram of OSR1 expression rate in ovarian cancer group and normal ovarian group(****P < 0.0001); D KM survival curve analysis for OSR1 expression in 86 patients with OC(Log Rank P = 0.02)

Fig. 2

OSR1 inhibited the NF-κB pathway. A–B Protein and mRNA expression levels of OSR1 in HOSEpiC and OC cells, including SKOV3, OVCAR3, COC1, and A2780; C–D Results of OSR1 protein and mRNA expression levels in SKOV3 and OVCAR3 cells with overexpressed OSR1; E–F Results of OSR1 protein and mRNA levels in A2780 cells transfected with OSR1-siRNA; G WB determined the p-IκBα, IκBα, p65, and p-p65 protein expression in OSR1 overexpressed SKOV3 and OVCAR3 cells; H WB determined the p-IκBα, IκBα, p65, and p-p65 protein expression in OSR1 knockdown A2780 cells. *P < 0.05; **P < 0.01;***P < 0.001;****P < 0.0001. Data shown represent the mean ± SD from three independent experiments rom three independent experiments, each performed in triplicate

Fig. 3

The effects of double inhibition of OSR1 and NF-κB pathway on the proliferation and the cell cycle of OC cells. A CCK-8 assay demonstrated that OSR1 knockdown promoted OC cell growth activity, which could be reversed by BAY 11-7082. B Flow cytometry assay suggested OSR1 knockdown promoted OC cell cycle progression, which could be reversed by BAY 11-7082. C WB determined the expression of PCNA and cyclinD1 after OSR1 knockdown or double inhibition of OSR1 and NF-κB pathway. **P < 0.01;***P < 0.001;****P < 0.0001. Data shown represent the mean ± SD from three independent experiments rom three independent experiments, each performed in triplicate

Fig. 4

The effects of double inhibition of OSR1 and NF-κB pathway on the invasion and migration of OC cells. A The wound healing assay demonstrated that OSR1 knockdown promoted OC migration in A2780 cells, which was reversed by BAY 11-7082 (× 100). B Transwell assay demonstrated that OSR1 knockdown suppressed invasion of OC cells, which was reversed by BAY 11-7082 (× 200). C ELISA assay detected the MMP-9 and MMP-2 expression in the four groups. *P < 0.05; ****P < 0.0001. Data shown represent the mean ± SD from three independent experiments rom three independent experiments, each performed in triplicate

Fig. 5

The influences of double inhibition of OSR1 and NF-κB pathway on the apoptosis of OC cells. A Annexin V-FITC/PI flow cytometry assay demonstrated that OSR1 knockdown inhibited OC apoptosis, which was reversed by BAY 11-7082. B The apoptotic cells detected by hoechst staining assay reduced significantly in A2780 OC cells with OSR1 knockdown but increased when treated the cells with BAY 11-7082. C WB detected the Bax, Bcl-2, Caspase-3, Cleaved Caspase-3 protein expression in the four groups. *P < 0.05;**, P < 0.01;***P < 0.001;****P < 0.0001. Data shown represent the mean ± SD from three independent experiments rom three independent experiments, each performed in triplicate