Long non‑coding RNA NEAT1 promotes ovarian cancer cell invasion and migration by interacting with miR‑1321 and regulating tight junction protein 3 expression

Abstract

Previous studies have reported that long non‑coding RNAs (lncRNAs) have a significant role in the metastasis of tumors, including ovarian cancer (OC). The aim of the present study was to demonstrate the function and working mechanism of lncRNA nuclear enriched abundant transcript 1 (NEAT1) in OC. The expressions of NEAT1 in OC were measured by reverse transcription‑quantitativePCR (RT‑qPCR). The effects of NEAT1 on cell proliferation, invasion, migration and epithelial‑mesenchymal transition (EMT) were detected by Cell Counting Kit‑8, transwell and wound healing assays, and western blotting. Dual‑luciferase reporter assays were performed to confirm the correlated between NEAT and miR‑1321, miR‑1321 and TJP3. The effect of NEAT1 on miR‑1321 and TJP3 was confirmed by RT‑qPCR and western blotting. Elevated expression of NEAT1 was observed in OC cell lines, and NEAT1 expression was found to be positively related to the expression of tight junction protein 3 (TJP3), which is important in cancer development. Moreover, the present results indicated that NEAT1 and TJP3 expression levels were negatively correlated with microRNA (miR)‑1321 expression in OC. Knockdown of NEAT1 attenuated the migration and invasion of OC cells, as well as increased miR‑1321 expression and in turn led to the reduction of TJP3. Thus, the present study demonstrated that NEAT1 regulates TJP3 expression by sponging miR‑1321 and enhances the epithelial‑mesenchymal transition, invasion and migration of OC cells. Overall, the present study identified the function and mechanism of NEAT1 in OC, suggesting that NEAT1 may be a promising therapeutic target for OC metastasis.

Figure 1.

NEAT1 expression and function in OC. (A) NEAT1 expression in normal epithelial cells (IOSE80) and OC cells (SKOV3, OVCAR-3, ES-2 and A2780) and (B) transfection efficiency of si-NEAT1 in ES-2 and A2780 cells were detected using reverse transcription-quantitative PCR. (C) Proliferative abilities of ES-2 and A2780 cells were measured using Cell Counting Kit-8 assays. (D) E-cadherin, N-cadherin and Vimentin protein expression level in ES-2 and A2780 cells were assessed using western blotting. (E) Migration and invasive abilities of ES-2 and A2780 cells were measured using Transwell assays. Scale bar, 100 µm. (F) Migration abilities of ES-2 and A2780 cells were measured using wound-healing assays. Scale bar, 100 µm. All data are representative of ≥3 independent experiments. Data are presented as the mean ± SD. *P<0.05 vs. control group; **P<0.01 vs. control group; ***P<0.001 vs. control group, as determined by Student's t-tests or ANOVA followed by a Tukey's post hos test. OC, ovarian cancer; NEAT1, nuclear enriched abundant transcript1; si, small interfering RNA; NC, negative control; OD, optical density.

Figure 2.

Interrelation of NEAT1, miR-1321 and TJP3. (A) StarBase website predicted the complementary binding sites between NEAT1 and miR-1321. (B) Transfection efficiency of miR-1321 mimics was detected using RT-qPCR. (C) Targeting relationship of NEAT1 and miR-1321 was identified using dual-luciferase reporter gene assays. (D) StarBase website predicted the complementary binding site between miR-1321 and TJP3. (E) Targeting relationship of miR-1321 and TJP3 was identified using dual-luciferase reporter gene assays. (F) Expression of miR-1321 in ES-2 and A2780 cells was measured using RT-qPCR. (G) Effect of NEAT1 on miR-1321 expression was determined using RT-qPCR. (H) Effect of miR-1321 on expression of TJP3 protein was measured using western blotting. Correlations of (I) lncRNA NEAT1 and miR-1321, (J) miR-1321 and TJP3, and (K) lncRNA NEAT1 and TJP3 on ovarian cancer specimens were investigated using a Pearson Correlation Coefficient. Data are representative of ≥3 independent experiments. Data are presented as the mean ± SD. *P<0.05 vs. control group; **P<0.01 vs. control group as determined by Student's t-tests or ANOVA followed by a Tukey's post hos test. NEAT1, nuclear enriched abundant transcript1; TJP3, tight junction protein 3; miR, microRNA; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR; lncRNA, long non-coding RNA; NC, negative control; WT, wild-type; MUT, mutant; si, small interfering RNA.

Figure 3.

TJP3 expression and function in OC. (A) TJP3 protein expression level in normal epithelial cells (IOSE80) and OC cells (SKOV3, OVCAR-3, ES-2 and A2780) were measured using western blotting. (B) Transfection efficiency of si-TJP3 in ES-2 and A2780 cells were detected using western blotting. (C) Proliferative abilities of ES-2 and A2780 cells were measured using Cell Counting Kit-8 assays. (D) E-cadherin, N-cadherin and Vimentin protein expression levels in ES-2 and A2780 cells were assessed using western blotting. (E) Migratory and invasive abilities of ES-2 and A2780 cells were measured using Transwell assays. Scale bar, 100 µm. (F) Migratory abilities of ES-2 and A2780 cells were measured using a wound-healing assay. Scale bar, 100 µm. All data are representative of ≥3 independent experiments. Data are presented as the mean ± SD. *P<0.05 vs. control group; **P<0.01 vs. control group as determined by Student's t-tests or ANOVA followed by a Tukey's post hos test. TJP3, tight junction protein 3; si, small interfering RNA; NC, negative control; OC, ovarian cancer.

Figure 4.

NEAT1 promotes cell epithelial-mesenchymal transition, migration and invasion of ovarian cancer by regulating TJP3. (A) Transfection efficiency of si-NEAT1 in ES-2 and A2780 cells were detected using reverse transcription-quantitative PCR. (B) Effect of NEAT1 on TJP3 protein expression was measured using western blotting. (C) Proliferative abilities of ES-2 and A2780 cells were measured using Cell Counting Kit-8 assays. (D) E-cadherin, N-cadherin and Vimentin protein expression levels in ES-2 and A2780 cells were assessed using western blotting. (E) Migratory and invasive abilities of ES-2 and A2780 cells were measured using Transwell assays. Scale bar, 100 µm. (F) Migratory abilities of ES-2 and A2780 cells were measured using a wound-healing assay. Scale bar, 100 µm. All data are representative of ≥3 independent experiments. Data are presented as the mean ± SD. *P<0.05 vs. NC group; **P<0.01 vs. NC group; ^#P<0.05 vs. si-NEAT1 treatment group; ^##P<0.01 vs. si-NEAT1 treatment group; ^###P<0.001 vs. si-NEAT1 treatment group as determined by Student's t-test or ANOVA followed by a Tukey's post hos test. TJP3, tight junction protein 3; NEAT1, nuclear enriched abundant transcript1; si, small interfering RNA; OE, overexpressed.