FEZF1-AS1 is a key regulator of cell cycle, epithelial-mesenchymal transition and Wnt/β-catenin signaling in nasopharyngeal carcinoma cells

Abstract

Background: Accumulating studies discloses that long non-coding RNAs (lncRNAs) serve important roles in human tumorigenesis, including nasopharyngeal carcinoma (NPC). The purpose of the present study was to determine the role of lncRNA FEZF1-AS1 in NPC.

Materials and methods: The expression levels of FEZF1-AS1 in NPC tissues and cell lines were detected by RT-qPCR analysis. MTT assay was performed to investigate the proliferation of NPC cells in vitro, whereas the migration and invasion of NPC cells were determined by wound healing assay and transwell assay. A nude mouse tumor model was established to study the role of FEZF1-AS1 in NPC tumorigenesis in vivo The expression levels of proteins were detected by Western blot assay.

Results: The results showed that FEZF1-AS1 expression was increased in the NPC tissues and cell lines, and the higher expression of FEZF1-AS1 was closely associated with poor prognosis of NPC patients. We further observed that knockdown of FEZF1-AS1 inhibited the proliferation of NPC cells in vitro and suppressed NPC xenograft growth in vivo through inducing G2/M cell cycle arrest. The migratory and invasive abilities of NPC cells were also reduced upon FEZF1-AS1 knockdown. Moreover, we demonstrated that inhibition of FEZF1-AS1 remarkably suppressed epithelial-mesenchymal transition (EMT) and reduced β-catenin accumulation in nucleus in NPC cells.

Conclusions: Collectively, we showed that FEZF1-AS1 might be a key regulator of cell cycle, EMT and Wnt/β-catenin signaling in NPC cells, which may be helpful for understanding of pathogenesis of NPC.

Keywords: EMT; FEZF1-AS1; Wnt/β-catenin signaling; cell cycle; nasopharyngeal carcinoma.

Figure 1. FEZF1-AS1 is up-regulated in NPC tissues and cell lines

(A) RT-qPCR analysis of FEZF1-AS1 expression in the NPC tissues and normal nasopharyngeal epithelial tissues. (B and C) Kaplan–Meier analysis of overall survival and disease-free survival in 71 NPC patients according to FEZF1-AS1 expression. (D) RT-qPCR analysis of FEZF1-AS1 expression in a panel of NPC cell lines. Data are presented as the mean ± SD from three independent experiments in vitro. *P<0.05 versus NP69 cells.

Figure 2. FEZF1-AS1 promotes NPC cell proliferation and cell cycle arrest

(A) The transfection efficacy was verified by RT-qPCR analysis. (B) The proliferation of 5-8F and HNE1 cells after transfection was detected by MTT assay. (C) The cell cycle distribution of 5-8F and HNE1 cells after transfection was detected by flow cytometric analysis. (D) The expression levels of p21 and Cyclin D1 in 5-8F and HNE1 cells after transfection were detected by Western blot analysis. Data are presented as the mean ± SD from three independent experiments in vitro. *P<0.05 versus si-NC-transfected 5-8F cells or empty vector-transfected HNE1 cells.

Figure 3. Knockdown of FEZF1-AS1 suppresses NPC tumorigenesis in vivo

(A) Tumor volume growth curves were plotted. (B) The average tumor weight in each group was shown. (C) RT-qPCR analysis of FEZF1-AS1 expression in the xenograft tissues. (D) The expression levels of p21 and Cyclin D1 in the xenograft tissues were detected by Western blot analysis. Data are presented as the mean ± SD (n = 5/group). *P<0.05 versus sh-NC group.

Figure 4. FEZF1-AS1 promotes NPC cell migration and invasion

(A) The migratory abilities of 5-8F and HNE1 cells after transfection were detected by wound healing assay. (B) The migration and invasion of 5-8F and HNE1 cells after transfection were detected by transwell assay. Data are presented as the mean ± SD from three independent experiments in vitro. *P<0.05 versus si-NC-transfected 5-8F cells or empty vector-transfected HNE1 cells.

Figure 5. FEZF1-AS1 induces EMT in NPC cells

The expression levels of E-cadherin, N-cadherin and Vimentin in 5-8F and HNE1 cells after transfection were detected by Western blot analysis. Data are presented as the mean ± SD from three independent experiments in vitro. *P<0.05 versus si-NC-transfected 5-8F cells or empty vector-transfected HNE1 cells.

Figure 6. FEZF1-AS1 activates Wnt/β-catenin signaling in NPC cells

(A) Dual luciferase reporter assay using TOP/FOP flash vectors was performed to determine the activity of Wnt/β-catenin signaling in 5-8F and HNE1 cells after transfection. (B) The expression levels of β-catenin in the nuclear and cytosolic fractions of 5-8F and HNE1 cells after transfection were detected by Western blot analysis. Data are presented as the mean ± SD from three independent experiments in vitro. *P<0.05 versus si-NC-transfected 5-8F cells or empty vector-transfected HNE1 cells.