Resveratrol Suppresses Human Nasopharyngeal Carcinoma Cell Growth Via Inhibiting Differentiation Antagonizing Non-Protein Coding RNA (DANCR) Expression

Abstract

BACKGROUND Although resveratrol has been found to show anti-cancer effects and potential chemotherapeutic activities in several cancers, the role and molecular mechanisms of resveratrol in nasopharyngeal carcinoma (NPC) remains poorly understood. This study aimed to investigate the effect of resveratrol in NPC progression and its molecular mechanism. MATERIAL AND METHODS Quantitative real-time polymerase chain reaction and western blotting were used to detect the expression of DANCR and PTEN. MTT assay and EdU assay were performed to detect the cell proliferation in NPC cells with different treatment. The effect of resveratrol on cell migration was explored by Transwell migration assay. RNA immunoprecipitation assay and chromatin immunoprecipitation assay were performed to test the interaction between DANCR, EZH2, and PTEN. A mouse xenograft model of NPC cell was established, and immunohistochemistry assay was performed to detect the PTEN expression. RESULTS Resveratrol treatment inhibited NPC cell growth and migration in a dose-dependent manner. Additionally, resveratrol downregulated the expression of DANCR and DANCR overexpressing abrogated the inhibition effect of resveratrol on NPC cell migration. Mechanistically, DANCR could bind to EZH2 and downregulated PTEN expression through mediating the binding of EZH2 on PTEN promoter. Furthermore, rescue experiments suggested resveratrol inhibited NPC cell growth and migration by the DANCR/PTEN pathway. Resveratrol significantly decreased the tumor volume and tumor weight and increased the expression of PTEN. CONCLUSIONS Resveratrol increased PTEN expression and suppressed NPC cell growth and migration through downregulation of DANCR.

Figure 1

Resveratrol treatment inhibited NPC cell growth and migration. SUNE-1 cells and 5–8F cells were treated with different concentration of resveratrol for 24 hours or 48 hours. (A) Cell growth and (B) migration were detected by MTT assay and Transwell migration assay, respectively. Data were shown as the mean±SD of 3 independent experiments. * P<0.05 compared with control. NPC – nasopharyngeal carcinoma; MTT – 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); SD – standard deviation.

Figure 2

DANCR mediated the anti-cancer effect of resveratrol in NPC. Relative DANCR level was determined by qRT-PCR in SUNE-1 cells and 5–8F cells treated with resveratrol (A) or transfected with pcDNA-DANCR or si-DANCR (B). (C) Cell migration was measured in SUNE-1 cells and 5–8F cells treated with resveratrol and pcDNA-DANCR. The protein level of PTEN was detected by western blot in SUNE-1 cells and 5–8F cells treated with resveratrol (D) or transfected with pcDNA-DANCR (E). Data were shown as the mean±SD of 3 independent experiments. * P<0.05 compared with control group or pcDNA group or si-NC group. DANCR – differentiation antagonizing non-protein coding RNA; NPC – nasopharyngeal carcinoma; qRT-PCR – quantitative real-time polymerase chain reaction; pcDNA – plasmid cloned DNA; si-DANCR – specific small interfering RNA (siRNA) targeting DANCR; si-NC – scrambled negative control; SD – standard deviation.

Figure 3

DANCR was required for EZH2 binding on PTEN promoter. (A) The expression of PTEN in SUNE-1 cells and 5–8F cells transfected with si-DANCR was determined by qRT-PCR and western blot analysis. (B) RIP assay was performed in SUNE-1 cells and 5–8F cells. (C) The expression of EZH2 in SUNE-1 cells and 5–8F cells transfected with si-EZH2. (D) The expression of PTEN in SUNE-1 cells and 5–8F cells transfected with si-EZH2 or si-PTEN was determined by qRT-PCR and western blot analysis. (E) ChIP analysis on PTEN promoter was performed in SUNE-1 cells and 5–8F cells transfected with si-DANCR. Data were shown as the mean±SD of 3 independent experiments. * P<0.05 versus si-NC group or IgG group. DANCR – differentiation antagonizing non-protein coding RNA; PTEN – phosphatase and tensin homolog; si-DANCR – specific small interfering RNA (siRNA) targeting DANCR; qRT-PCR – quantitative real-time polymerase chain reaction; RIP – RNA immunoprecipitation; ChIP – chromatin immunoprecipitation; SD – standard deviation, si-NC – scrambled negative control.

Figure 4

Resveratrol inhibited NPC cell growth and migration by DANCR/PTEN pathway. SUNE-1 cells and 5–8F cells were treated with resveratrol and transfected with pcDNA-DANCR. (A) The expression of PTEN was determined by qRT-PCR and western blot analysis in these cells. (B) EdU incorporation assay and (C)Transwell migration assay was performed in these cells. Data were shown as the mean±SD of 3 independent experiments. * P<0.05 versus control group; ^# P<0.05 versus RSV+pcDNA group. NPC – nasopharyngeal carcinoma; DANCR – differentiation antagonizing non-protein coding RNA; PTEN – phosphatase and tensin homolog; qRT-PCR – quantitative real-time polymerase chain reaction; EdU – 5-ethynyl-2′-deoxyuridine; SD – standard deviation; RSV – resveratrol; pcDNA – plasmid cloned DNA.

Figure 5

Resveratrol suppressed NPC tumor growth in vivo. (A) The tumor volume was calculated every 4 days. (B) The tumor weight was measured. (C) Relative DANCR level in tumor tissue was detected by qRT-PCR. (D) Representative images of tumors in each group; bar=1 cm. (E) Immunohistochemical assay was performed using PTEN antibody in tumor tissues. Data were shown as the mean ± SD of 3 independent experiments. * P<0.05 versus control group. NPC – nasopharyngeal carcinoma; DANCR – differentiation antagonizing non-protein coding RNA; qRT-PCR – quantitative real-time polymerase chain reaction; PTEN – phosphatase and tensin homolog; SD – standard deviation.