Repression of lncRNA PART1 attenuates ovarian cancer cell viability, migration and invasion through the miR-503-5p/FOXK1 axis

Abstract

Background: Ovarian cancer (OC) is a female malignant tumor with a high fatality rate. Long non-coding RNAs (lncRNAs) are deeply involved in OC progression. The aim of this study is to explore the specific mechanism of lncRNA prostate androgen-regulated transcript 1 (PART1) in OC.

Methods: Quantitative real time PCR was utilized to determine the expression levels of PART1, microRNA (miR)-503-5p and forkhead-box k1 (FOXK1) in OC tissues and/or cells. The cell viability, migration, and invasion in OC were evaluated by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-h-tetrazolium bromide assay, wound healing assay and transwell invasion assay, respectively. Flow cytometry was used to analyze the cell apoptosis. The xenograft tumor was conducted in nude mice to verify the effect of PART1 knockdown on OC in vivo. The target relationships among PART1, miR-503-5p and FOXK1 were predicted by StarBase, and verified by luciferase reporter assay. The level of FOXK1 was assessed by western blot.

Results: Increased expression of PART1 and FOXK1 was observed in OC tissues or cells, whereas miR-503-5p was downregulated. PART1 silencing or miR-503-5p overexpression repressed the cell viability, migration and invasion, and protomed apoptosis. Meanwhile, miR-503-5p was a target of PART1, and FOXK1 was a direct target gene of miR-503-5p. Both downregulation of miR-503-5p and upregulation of FOXK1 partly relieved the suppressive effects of PART1 knockdown on the oncogenicity of OC in vitro.

Conclusion: Decreased PART1 represses the cell viability, migration and invasion of OC via regulating the miR-503-5p/FOXK1 axis, which provided an underlying target for treating OC.

Keywords: FOXK1; Ovarian cancer; lncRNA PART1; miR-503-5p.

Fig. 1

High expression of PART1 is identified in OC tissues and cell lines. A The expression of PART1 in different types of human cancer. B The expression of PART1 in OC tissues (n = 50) and normal ovary epithelial tissues (n = 50) and C in III/IV stage tissues (n = 28) and I/II stage tissues (n = 22) was detected by qRT-PCR. **P < 0.01 vs. the I/II group. D The expression of PART1 in metastasis tissues (n = 23) and non-metastasis tissues (n = 27) was detected. **P < 0.01 vs. the non-metastasis group. E The expression of PART1 in OC cell lines (CaoV-3, SK-OV-3, and HO-8910) and human ovarian epithelial cells (IOSE80) was detected by qRT-PCR. **P < 0.01, ***P < 0.001 vs. the IOSE8 cells group

Fig. 2

PART1 knockdown represses the viability, migration, invasion and induces apoptosis of OC cell lines. A The expression of PART1 after transfection of sh-PART1–1/− 2/− 3/NC into OC cells (SK-OV-3 and HO-8910) was detected by qRT-PCR. B-E The viability, migration, invasion, and apoptosis of OC cells transfected with sh-PART1–2/NC were measured by MTT assay, wound healing assay, invasion assay, and flow cytometry, respectively. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the sh-NC group

Fig. 3

PART1 targets miR-503-5p. A The predicted complementary binding site of PART1 and miR-503-5p. B The luciferase activity in OC cells co-transfected with pGL3-PART1 WT/pGL3-PART1 MUT and miR-503-5p/miR-NC was determined by dual luciferase reporter assay. ***P < 0.001 vs. the miR-NC group. C The expression of miR-503-5p in OC tissues (n = 50) and normal tissues (n = 50) was detected by qRT-PCR. ***P < 0.001 vs. the normal group. D The correlation between the expression PART1 and miR-503-5p in OC tissues was assessed by Pearson’s correlation analysis. P < 0.001, R² = 0.665. E The expression of miR-503-5p after transfection of sh-PART1–2/NC into OC cells was detected by qRT-PCR. ***P < 0.001 vs. the sh-NC group

Fig. 4

Overexpressed miR-503-5p inhibits the viability, migration and invasion, and induces apoptosis of SK-OV-3 cells. A The expression of miR-503-5p after transfection of miR-NC/ miR-503-5p mimics or inhibitor NC/miR-503-5p inhibitor into SK-OV-3 cells was detected by qRT-PCR. ***P < 0.001 vs. the miR-NC group, ###P < 0.001 vs. the inhibitor NC group. B-E The viability, migration, invasion and apoptosis of SK-OV-3 cells transfected with miR-503-5p mimics/miR-NC was measured by MTT assay, wound healing assay, invasion assay and flow cytometry, respectively. **P < 0.01 vs. the miR-NC group

Fig. 5

miR-503-5p targets and mediates FOXK1 expression. A The predicted complementary binding site of miR-503-5p and FOXK1. B The luciferase activity in OC cells was determined by dual luciferase reporter assay. ***P < 0.001 vs. the miR-NC group. C The expression of FOXK1 in OC tissues (n = 50) and normal tissues (n = 50) was detected by qRT-PCR. ***P < 0.001 vs. the normal group. D-E The correlation between the expression miR-503-5p and FOXK1 and E PART1 and FOXK1 in OC tissues was assessed by Pearson’s correlation analysis. P < 0.0001, R² = 0.6138. F The protein level of FOXK1 in OC cells after transfection of miR-503-5p mimics/miR-NC or sh-PART1–2/NC (G) was determined by western blot assay. H The expression of FOXK1 in SK-OV-3 cells transfected with sh-PART1–2/NC was detected by qRT-PCR. **P < 0.01 vs. the sh-NC group. I The apoptosis of SK-OV-3 cells transfected with sh-PART1–2/NC was analyzed by flow cytometry. **P < 0.01 vs. the sh-NC group

Fig. 6

FOXK1 overexpression reverses the ameliorative malignant behaviors of OC caused by PART1 knockdown. A The expression of PART1 after transfection of pcDNA3.1-FOXK1/pcDNA3.1 into SK-OV-3 cells was detected by qRT-PCR. ***P < 0.001 vs. the pcDNA3.1 group. B-D The viability, migration, and invasion of SK-OV-3 cells transfected with different plasmids was measured by MTT assay, wound healing assay, and transwell invasion assay. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the sh-NC + pcDNA3.1-NC group; ##P < 0.01, ###P < 0.001 vs. the sh-PART1–2 + pcDNA3.1-NC group

Fig. 7

Silencing of PART1 suppresses the growth of tumor xenograft in vivo. A The image of solid tumor after injection of sh-PART1–2/sh-NC. B The tumor volume and C tumor weight after injection of sh-PART1–2/sh-NC. D The expression of PART1, E miR-503-5p, and FOXK1 (F) in tumor xenograft tissues injected with sh-PART1–2/sh-NC was detected by qRT-PCR and Western blot. G TUNEL assay was used to detect the TUNEL positive cells in mice. **P < 0.01, ***P < 0.001 vs. the sh-NC group