NEAT1 Overexpression Indicates a Poor Prognosis and Induces Chemotherapy Resistance via the miR-491-5p/ SOX3 Signaling Pathway in Ovarian Cancer

Abstract

Background: Accumulated studies have reported that dysregulated long non-coding RNAs (lncRNAs) are crucial in ovarian cancer (OC) initiation and development. However, detailed biological functions of lncRNA NEAT1 during the progression of OC remains to be uncovered.

Purpose: Our aim was to identify the role of NEAT1 in cisplatin resistance of ovarian cancer and the underlying mechanisms.

Methods: The expression patterns of NEAT1 in OC cell lines and tissue samples were identified by qRT-PCR. The cisplatin (DDP) sensitivity of OC cells was detected by MTT and CCK8 assay, while OC cell apoptosis and cell cycle were detected using flow cytometer assays. In addition, effects of NEAT1 on tumor growth were determined by xenograft tumor model. Luciferase reporter assay was conducted to prove the regulatory relation of miR-491-5p, NEAT1, and SOX3. Importantly, the expression of NEAT1 in exosomes from cisplatin-resistant patients was also determined by using qRT-PCR.

Results: In this study, upregulated NEAT1 was detected in OC cell lines and tissues. Meanwhile, NEAT1 was also increased in cisplatin-resistant OC cell lines and tissues. Upregulation of NEAT1 inhibited cisplatin-induced OC cell apoptosis and promoted cell proliferation, while knockdown of NEAT1 played the opposite role. These effects were also observed in vivo. Furthermore, direct interaction was observed between NEAT1 and miR-491-5p. NEAT1 led to the upregulation of miR-491-5p-targeted SOX3 mRNA. Importantly, this study also showed upregulated NEAT1 expression in serum exosomes derived from cisplatin-resistant patients.

Conclusion: NEAT1 is vital in the chemoresistance of ovarian cancer through regulating miR-491-5p/SOX3 pathway, showing that NEAT1 might be a potential target for OC resistance treatment.

Keywords: NEAT1; SOX3; chemoresistance; exosomes; miR-491-5p; ovarian cancer.

FIGURE 1

NEAT1 was upregulated in cisplatin-resistant OC. (A) QRT-PCR analysis of NEAT1 expressions in OC or adjacent normal tissues. (B) Relative NEAT1 expression in cisplatin-resistant OC and cisplatin-sensitive OC. (C) Kaplan-Meier curve of the estimated overall survival of both low and high NEAT1 expression groups. (D,E) Relative NEAT1 expression in both cisplatin-resistant and cisplatin-sensitive OC cell lines. Data were reported as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2

NEAT1 affected the OC cell proliferation and apoptosis in vitro. (A) QRT-PCR analysis of NEAT1 expression in OC cells post transfection with si-NEAT1 or NEAT1 plasmid. (B) IC50 of cisplatin-resistant or cisplatin-sensitive cells with increased or decreased NEAT1 level. (C) Promotion effects of overexpressed NEAT1 on the proliferation of A2780 and HO8910 cells during CCK-8 assay. (D) Suppression of NEAT1 silencing on the proliferation of A2780/DDP and HO8910 cells with 4 mg/mL cisplatin as shown by CCK-8 assay. (E–H) Flow cytometry of apoptosis of A2780 and HO8910 cells post transfection with vector or pcDNA-NEAT1; A2780/DDP and HO8910/DDP cells post transfection si-Control or si-NEAT1 before the stimulation of 4 mg/mL cisplatin treatments. All tests were conducted for at least three times. Data were reported as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3

lncRNA NEAT1 promoted cisplatin resistance in vivo. (A) Photos of tumors in xenograft-transplanted nude mouse tumor models after the treatment of oral cisplatin (qd) or NS as control for 31 days in different groups. (B) Measurements of tumor volumes every 4 days using calipers. (C) Weights of tumors in xenografts of different groups. (D) Representative images of the mesenteric membrane of six mice per group are shown. *Indicates tumor nodule. (E) IHC analysis of Ki-67 expression in xenografts of different groups. All tests were conducted for at least three times. Data were reported as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4

NEAT1 functioned as a molecular sponge of miR-491-5p in OC cells. (A) Decrease in miR-491-5p expression by overexpressed NEAT1. (B) Increase in miR-491-5p expression by silencing NEAT1. (C) Relative miR-491-5p expression in cisplatin-sensitive and cisplatin-resistant OC groups. (D) Relative miR-491-5p expression in a set of OC cell lines. (E,F) Proliferation of si-NEAT1-treated cells decreased and rescued by miR-491-5p inhibitor during CCK8 assay. (G) Sequence of NEAT1 containing highly conserved putative miR-491-binding sites as shown by StarBase v.2.0. (H) Luciferase activity of NEAT1-WT luciferase reporter vector remarkably reduced by miR-491-5p mimic relative to NC. All tests were conducted for at least three times. Data were reported as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5

SOX3 was a direct target of miR-491-5p. (A) Binding sites between SOX3 and miR-491-5p predicted by bioinformatics analysis. (B) Luciferase activity of SOX3-WT in OC cells considerably reduced by miR-491-5p mimics as indicated by luciferase reporter assay. (C) Relative SOX3 expression in OC patients. (D) Relative SOX3 expression in cancer cell lines. (E,F) Real-time PCR analysis and Western Blot assay for determining SOX3 expression in cells with rescue assay. All tests were conducted for at least three times. Data were reported as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6

NEAT1 was secreted into exosomes from OC patients’ sera. (A) Typical electron microscope image of exosomes from OC patients’ sera. (B) Western blotting for biomarkers of exosomes from purified serum exosomes. (C) QRT-PCR for the abundance of NEAT1 in serum exosomes, and NEAT1 levels in sera. (D) Negative association between NEAT1 and miR-491-5p expressions in exosomes extracted from OC patients’ sera. **p < 0.01.