LncRNA NEAT1 contributes to paclitaxel resistance of ovarian cancer cells by regulating ZEB1 expression via miR-194

Abstract

Background: Chemoresistance is one of the major obstacles for cancer therapy in the clinic. Nuclear paraspeckle assembly transcript 1 (NEAT1) has been reported as an oncogene in most malignancies such as lung cancer, esophageal cancer, and gastric cancer. This study is designed to investigate the function of NEAT1 in paclitaxel (PTX) resistance of ovarian cancer and its potential molecular mechanism.

Patients and methods: The expressions of NEAT1 and miR-194 in ovarian cancer tissues and cells were estimated by quantitative real-time polymerase chain reaction (qRT-PCR). MTT, flow cytometry, and Western blot assays were used to assess the effect of NEAT1 on PTX resistance in PTX-resistant ovarian cancer cells. Luciferase reporter assay was applied to examine the association between NEAT1, zinc finger E-box-binding homeobox 1 (ZEB1) and miR-194. Xenograft tumor model was established to confirm the biological role of NEAT1 in PTX resistance of ovarian cancer in vivo.

Results: NEAT1 was upregulated, and miR-194 was downregulated in PTX-resistant ovarian cancer tissues and cells. Functionally, NEAT1 knockdown enhanced cell sensitivity to PTX via promoting PTX-induced apoptosis in vitro. NEAT1 was identified as a molecular sponge of miR-194 to upregulate ZEB1 expression. Mechanistically, NEAT1-knockdown-induced PTX sensitivity was mediated by miR-194/ZEB1 axis. Moreover, NEAT1 knockdown improved PTX sensitivity of ovarian cancer in vivo.

Conclusion: NEAT1 contributed to PTX resistance of ovarian cancer cells at least partly through upregulating ZEB1 expression by sponging miR-194, elucidating a novel regulatory pathway of chemoresistance in PTX-resistant ovarian cancer cells and providing a possible long noncoding RNA (lncRNA)-targeted therapy for ovarian cancer.

Keywords: PTX; ZEB1; ceRNAs; lncRNA NEAT1; miR-194.

Figure 1

Expressions of NEAT1 and miR-194 in PTX-resistant ovarian cancer tissues and cells. Notes: qRT-PCR analysis of NEAT1 (A) and miR-194 (B) expressions in 18 treatment-responsive patients and 14 treatment-resistant patients. qRT-PCR analysis of NEAT1 (C and D) and miR-194 (E and F) expressions in parental ovarian cancer cells (SKOV3 and HeyA-8) and PTX-resistant ovarian cancer cells (SKOV3/PTX and HeyA-8/PTX). *P<0.05. Abbreviations: NEAT1, nuclear paraspeckle assembly transcript 1; PTX, paclitaxel; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 2

NEAT1 knockdown-sensitized SKOV3/PTX and HeyA-8/PTX cells to PTX. Notes: IC₅₀ value of PTX in SKOV3 and SKOV3/PTX cells (A) as well as in HeyA-8 and HeyA-8/PTX cells (B). Expression of NEAT1 in si-NEAT1- or si-con-transfected SKOV3/PTX (C) and HeyA-8/PTX (D) cells was estimated by qRT-PCR. Protein levels of P-gp and GST-π were determined by Western blot in SKOV3/PTX (E) and HeyA-8/PTX (F) cells after introduction with si-NEAT1 or si-con. PTX resistance of si-NEAT1- or si-con-treated SKOV3/PTX (G) and HeyA-8/PTX (H) cells was assessed using IC₅₀ value of PTX by MTT assay. Apoptotic rate was measured by flow cytometry after SKOV3/PTX (I) and HeyA-8/PTX (J) cells transfected with si-NEAT1 or si-con were treated with PTX for 48 h. *P<0.05. Abbreviations: GST-π, glutathione S-transferase π; IC₅₀, the concentration of PTX causing 50% inhibition of growth; MTT, (4-5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NEAT1, nuclear paraspeckle assembly transcript 1; P-gp, P-glycoprotein; PTX, paclitaxel; qRT-PCR, quantitative real-time polymerase chain reaction; si-con, siRNA control.

Figure 2

NEAT1 knockdown-sensitized SKOV3/PTX and HeyA-8/PTX cells to PTX. Notes: IC₅₀ value of PTX in SKOV3 and SKOV3/PTX cells (A) as well as in HeyA-8 and HeyA-8/PTX cells (B). Expression of NEAT1 in si-NEAT1- or si-con-transfected SKOV3/PTX (C) and HeyA-8/PTX (D) cells was estimated by qRT-PCR. Protein levels of P-gp and GST-π were determined by Western blot in SKOV3/PTX (E) and HeyA-8/PTX (F) cells after introduction with si-NEAT1 or si-con. PTX resistance of si-NEAT1- or si-con-treated SKOV3/PTX (G) and HeyA-8/PTX (H) cells was assessed using IC₅₀ value of PTX by MTT assay. Apoptotic rate was measured by flow cytometry after SKOV3/PTX (I) and HeyA-8/PTX (J) cells transfected with si-NEAT1 or si-con were treated with PTX for 48 h. *P<0.05. Abbreviations: GST-π, glutathione S-transferase π; IC₅₀, the concentration of PTX causing 50% inhibition of growth; MTT, (4-5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NEAT1, nuclear paraspeckle assembly transcript 1; P-gp, P-glycoprotein; PTX, paclitaxel; qRT-PCR, quantitative real-time polymerase chain reaction; si-con, siRNA control.

Figure 3

NEAT1 inhibited miR-194 expression. Notes: (A) Graphical representation of predicted wild-type or mutated binding sequences in NEAT1. Luciferase reporter assay in SKOV3/PTX (B) and HeyA-8/PTX (C) cells after cotransfection with established luciferase reporter vectors (pGL3-NEAT1-Wt or pGL3-NEAT1-Mut) and miR-194 or miR-con. Expression of miR-194 was examined by qRT-PCR in SKOV3/PTX (D) and HeyA-8/PTX (E) cells treated with pcDNA-NEAT1, si-NEAT1, or respective controls. *P<0.05. Abbreviations: miR-con, miRNA control; NEAT1, nuclear paraspeckle assembly transcript 1; PTX, paclitaxel; qRT-PCR, quantitative real-time polymerase chain reaction; si-con, siRNA control; Wt, wild type; Mut, mutant.

Figure 4

NEAT1 knockdown increased PTX sensitivity in PTX-resistant ovarian cancer cells by repressing miR-194. Notes: SKOV3/PTX and HeyA-8/PTX cells were transfected with miR-194, si-NEAT1, si-NEAT1 + anti-miR-194, or matched controls. Protein levels of P-gp and GST-π in transfected SKOV3/PTX (A) and HeyA-8/PTX (B) cells were examined by Western blot. IC₅₀ value of PTX was measured by MTT assay after the transfected SKOV3/PTX (C) and HeyA-8/PTX (D) cells were treated with various doses of PTX for 48 h. Flow cytometry analysis was performed to detect apoptosis after transfected. SKOV3/PTX (E) and HeyA-8/PTX (F) cells were exposed to PTX for 48 h. *P<0.05. Abbreviations: GST-π, glutathione S-transferase π; IC₅₀, the concentration of PTX causing 50% inhibition of growth; MTT, (4-5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; miR-con, miRNA control; NEAT1, nuclear paraspeckle assembly transcript 1; P-gp, P-glycoprotein; PTX, paclitaxel; si-con, siRNA control.

Figure 5

NEAT1 upregulated ZEB1 expression via sponging miR-194. Notes: (A) Schematic representation of the predicted wild-type or mutant miR-194-binding sites in the 3′UTR sequences of ZEB1. The luciferase activity was detected by luciferase reporter assay in SKOV3/PTX (B) and HeyA-8/PTX (C) cells cotransfected with luciferase reporter plasmids (pGL3-ZEB1-3′UTR-Wt or pGL3-ZEB1-3′UTR-Mut) and miR-194, miR-194+ pcDNA-NEAT1, or corresponding control. Protein level of ZEB1 in SKOV3/PTX (D) and HeyA-8/PTX (E) cells introduced with si-NEAT1, miR-194, miR-194+ pcDNA-NEAT1, or matched controls was estimated by Western blot. *P<0.05. Abbreviations: miR-con, miRNA control; NEAT1, nuclear paraspeckle assembly transcript 1; PTX, paclitaxel; si-con, siRNA control; ZEB1, zinc finger E-box-binding homeobox 1; Wt, wild type; Mut, mutant.

Figure 6

ZEB1-knockdown-induced sensitivity of PTX was suppressed by reintroduction of pcDNA-NEAT1 or anti-miR-194. Notes: SKOV3/PTX and HeyA-8/PTX cells were transfected with si-ZEB1, si-ZEB1 + pcDNA-NEAT1, si-ZEB1 + anti-miR-194, or matched controls. Protein level of ZEB1 was determined by Western blot in transfected SKOV3/PTX (A) and HeyA-8/PTX (B) cells. PTX resistance in transfected SKOV3/PTX (C) and HeyA-8/PTX (D) cells was assessed using IC₅₀ value of PTX by MTT assay. Apoptotic rate in transfected SKOV3/PTX (E) and HeyA-8/PTX (F) cells with PTX treatment for 48 h was evaluated by flow cytometry analysis. *P<0.05. Abbreviations: IC₅₀, the concentration of PTX causing 50% inhibition of growth; miR-con, miRNA control; NEAT1, nuclear paraspeckle assembly transcript 1; PTX, paclitaxel; si-con, siRNA control; ZEB1, zinc finger E-box-binding homeobox 1.

Figure 7

NEAT1 knockdown enhanced PTX sensitivity of ovarian cancer cells in vivo. Notes: SKOV3/PTX cells introduced with sh-con or sh-NEAT1 were inoculated subcutaneously into the nude mice. The mice were given PBS or 3 mg/kg PTX (once every 3 days) via subcutaneous injection on day 6 after inoculation. (A) Growth curve of xenografted tumors. (B) Weights of resected tumor masses. (C) NEAT1 expression in xenografted tumors. *P<0.05. Abbreviations: NEAT1, nuclear paraspeckle assembly transcript 1; PTX, paclitaxel; sh, short hairpin; PBS, phosphate buffered solution.