The Long Noncoding RNA NEAT1 Targets miR-34a-5p and Drives Nasopharyngeal Carcinoma Progression via Wnt/β-Catenin Signaling

Abstract

Purpose: Long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) has been deemed an oncogene in many human cancers. However, the underlying mechanism of NEAT1 in nasopharyngeal carcinoma (NPC) progression remains largely unclear.

Materials and methods: Quantitative real-time PCR assay was performed to assess the expression of NEAT1 and miR-34a-5p in NPC tissues and cells. Western blot analysis was used to observe cell epithelial to mesenchymal transition (EMT) and the activation of Wnt/β-catenin signaling in 5-8F cells. MiRNA directly interacting with NEAT1 were verified by dual-luciferase reporter assay and RNA immunoprecipitation. Cell proliferation ability was determined by CCK-8 assay, and cell migration and invasion capacities were assessed by transwell assays. An animal model was used to investigate the regulatory effect of NEAT1 on tumor growth in vivo.

Results: Our data revealed that NEAT1 is upregulated, while miR-34a-5p is downregulated in NPC tissues and cell lines. NEAT1 knockdown repressed tumor growth in vitro and in vivo. Additionally, we discovered that NEAT1 directly binds to miR-34a-5p and suppresses miR-34a-5p expression. Moreover, NEAT1 knockdown exerted suppression effects on cell proliferation, migration, invasion, and EMT by miR-34a-5p. NEAT1 knockdown blocked Wnt/β-catenin signaling via miR-34a-5p.

Conclusion: Our study demonstrated that NEAT1 targets miR-34a-5p at least partly to drive NPC progression by regulating Wnt/β-catenin signaling, suggesting a potential therapeutic target for NPC.

Keywords: Nasopharyngeal carcinoma; Wnt/β-catenin signaling pathway; lncRNA NEAT1; miR-34a-5p.

Fig. 1. Upregulation of NEAT1 expression in NPC tissues and cell lines. (A and B) NEAT1 expression was detected by qRT-PCR assay in NPC tissues and corresponding normal tissues. (C) qRT-PCR assay of NEAT1 expression in nasopharyngeal epithelial cells (NP69) and NPC cell lines (CNE1, CNE2, SUNE1, 5-8F and SUNE2). (D) Kaplan-Meier survival method and log-rank test were performed to analyze the association between NEAT1 level and prognosis of NPC patients. NEAT1, nuclear paraspeckle assembly transcript 1; NPC, nasopharyngeal carcinoma.

Fig. 2. NEAT1 directly binds to miR-34a-5p and suppresses miR-34a-5p expression. (A) The predicted miR-34a-5p binding sequence within NEAT1 wild-type (WT) and a mutated form (MUT). 5-8F cells were cotransfected with NEAT1-WT or NEAT1-MUT and miR-NC mimics, miR-34a-5p mimics (B), in-miR-NC, and in-miR-34a-5p (C), followed by measurement of relative luciferase activity. (D) RIP experiment was performed using anti-Ago2 in 5-8F cells transfected with miR-34a-5p mimics and anti-IgG as a negative control, and Western blot analysis was used to validate successful RIP on the Enhanced Chemiluminescence Western blot system with ImageJ software. 5-8F cells were transfected with pcDNA, pcDNA-NEAT1, si-NC, or si-NEAT1, followed by detection of NEAT1 (E) and miR-34a-5p (F) expression levels. MiR-34a-5p expression was assessed in NPC tissues (G) and cell lines (H). NEAT1, nuclear paraspeckle assembly transcript 1; NPC, nasopharyngeal carcinoma; IP, immunoprecipitation complexes.

Fig. 3. si-NEAT1 inhibited NPC cell proliferation, migration, invasion, and EMT by miR-34a-5p. 5-8F cells were transfected with si-NC, si-NEAT1, si-NEAT1+in-miR-NC, and si-NEAT1+in-miR-34a-5p. (A) qRT-PCR assay of miR-34a-5p expression in treated cells. (B) Transwell assay of cell migration ability in treated cells. (C) Transwell assay of cell invasion capacity in treated cells. (D) CCK-8 assay of cell proliferation ability in treated cells. (E-H) Western blot analysis of E-cadherin, N-cadherin, and Vimentin expression levels in treated cells. Bands were quantified by ImageJ software. NEAT1, nuclear paraspeckle assembly transcript 1; NPC, nasopharyngeal carcinoma; EMT, epithelial to mesenchymal transition.

Fig. 4. si-NEAT1 blockaded the Wnt/β-catenin pathway by miR-34a-5p. 5-8F cells were transfected with si-NC, si-NEAT1, si-NEAT1+in-miR-NC, and si-NEAT1+in-miR-34a-5p (A), followed by measurement of β-catenin (B), cyclin D1 (C), and c-myc (D) expression levels by Western blot analysis. Bands were quantified by ImageJ software. NEAT1, nuclear paraspeckle assembly transcript 1.

Fig. 5. NEAT1 knockdown represses tumor growth and EMT by Wnt/β-catenin signaling in vivo. Nude mice were subcutaneously injected with 1.0×10⁷ 5-8F cells stably infected by sh-NC or sh-NEAT1, and the mice were euthanized at 5 weeks after implantation. (A) At 7 days after cell implantation, tumor volumes were measured every one week. (B) The average weight of tumors isolated from xenograft mice. qRT-PCR assay of NEAT1 (C) and miR-34a-5p (D) expression levels in excised tumors. Western blot analysis of E-cadherin, N-cadherin, and Vimentin protein levels (E), as well as β-catenin, cyclin D1, and c-myc expression levels (F), in excised tumor tissues with ImageJ software. NEAT1, nuclear paraspeckle assembly transcript 1; EMT, epithelial to mesenchymal transition.