NELFE promoted pancreatic cancer metastasis and the epithelial‑to‑mesenchymal transition by decreasing the stabilization of NDRG2 mRNA

Abstract

Negative elongation factor E (NELFE) has been demonstrated to promote cancer progression as an RNA‑binding protein (RBP). However, the expression patterns, biological role and molecular mechanism of NELFE in pancreatic cancer (PC) remain largely unknown. The expression levels of NELFE in 120 pairs of PC tissues and adjacent non‑tumor clinical samples collected from patients with PC were examined via reverse transcription‑quantitative (RT‑q) PCR and immunohistochemistry. The mRNA expression levels of NELFE, N‑Myc downstream‑regulated gene 2 (NDRG2), c‑Myc, survivin and cyclin D1 were detected via RT‑qPCR. The protein expression levels of NELFE, NDRG2, total β‑catenin, nuclear β‑catenin, cytosolic β‑catenin, E‑cadherin, N‑cadherin and Vimentin were measured by western blotting. NELFE and NDRG2 were then knocked‑down by short hairpin (sh)RNA. PC cell proliferation was detected by MTT and colony formation assays. Invasion and migration were detected by transwell assays. The interaction between NELFE and NDRG2 was detected by luciferase reporter assays, mRNA decay assays and RNA immunoprecipitation. NELFE expression was increased in PC tissues compared with the paired non‑cancerous tissues. NELFE expression was upregulated in PC cells when compared with normal pancreatic cells (HPDE6‑C7). The present study revealed that knockdown of NELFE inhibited the proliferation, invasion and migration of PC cells. In addition, transfection of the sh‑NELFE vector inhibited the epithelial‑to‑mesenchymal transition in PC cells by suppressing the expression and nuclear accumulation of β‑catenin. Further mechanistic studies revealed that NELFE activates the Wnt/β‑catenin signaling pathway by decreasing the stabilization of NDRG2 mRNA in PC. To the best of our knowledge, these results revealed the promotional function of NELFE on PC tumorigenesis and metastasis for the first time, helping to provide a promising strategy for the treatment of patients with PC.

Figure 1

Increased NELFE expression in PC tissues and cells. (A) The expression of NELFE in samples was analyzed by reverse transcription-quantitative PCR. (B) Analysis of the expression of NELFE in samples using an immunohistochemistry assay. The positive expression of NELFE was brown and yellow. The nuclei of cells were blue. (C) NELFE expression levels in various human PC cell lines and HPDE6-C7 cells. (D) Comparison of Overall survival between PC patients with high and low NELFE expression levels (n=120). The 120 pair of PC tissues and adjacent non-tumor clinical samples were removed from patients with PCs. *P<0.05 vs. HPDE6-C7; ^**P<0.01 vs. non-cancerous tissues or NELFE-low group. NELFE, negative elongation factor E; PC, pancreatic cancer.

Figure 2

NELFE promoted the malignant biological behavior of PC cells. (A) The mRNA expression of NELFE was decreased following transfection of the NELFE-shRNA vector in PaCa-2 and PANC-1 cells. (B) The protein expression of NELFE was decreased following transfection of the NELFE-shRNA vector in PaCa-2 and PANC-1 cells. (C) MTT assay demonstrated that decreased NELFE suppressed PC cell growth. (D) The decreased NELFE inhibited clone formation of PC cells. (E) Transwell assay revealed that knockdown of NELFE inhibited PC cell invasion and migration. The invaded cells were quantified by counting the cells in 10 randomly selected fields (×200 magnification). All experiments were repeated in triplicate. ^*P<0.05, ^**P<0.01. NELFE, negative elongation factor E; PC, pancreatic cancer; shRNA, short hairpin RNA; NC, negative control.

Figure 3

NELFE promoted EMT and Wnt/β-catenin signaling way in PC. (A) Western blot analysis of EMT-associated protein and β-catenin expression after the downregulation of NELFE in PC cells. GAPDH was used as an internal control. (B) Downregulation of NELFE in PC cells reduced the nuclear accumulation of β-catenin. (C) The dual luciferase reporter assay showed that decreased NELFE inhibited the transactivation of the TCF reporter in PC cells. (D) Reverse transcription-quantitative PCR assay revealed that the expression of the Wnt/β-catenin downstream genes (c-Myc, Survivin and cyclinD1) was decreased due to the knockdown of NFLFE in PC cells. All experiments were repeated three times. ^*P<0.05, ^**P<0.01 vs NC group. NELFE, negative elongation factor E; EMT, epithelial-to-mesenchymal transition; PC, pancreatic cancer; shRNA, short hairpin RNA; NC, negative control.

Figure 4

NDRG2 is an essential downstream effect of NELFE in PC. (A) NDRG2 expression was measured by western blotting in PC cells with NELFE knockdown. (B) The correlation between NELFE and NDRG2 mRNA expression in 120 samples from patients with PC was analyzed via person correlation analysis (R=−0.776). (C) Western blot analysis of associated protein and β-catenin expression following the downregulation of NDRG2 in PC cells with decreased NELFE. (D) Decreased NDRG2 enhanced the nuclear accumulation of β-catenin in PC cells with decreased NELFE. (E) MTT assay showed downregulation of NDRG2 rescued the function of sh-NELFE on PC cells proliferation. (F) The downregulation of NDRG2 rescued the function of decreased NELFE on PC cells colony formation ability. (G) Knockdown of NDRG2 rescued the inhibition function of decreased NELFE on PC cells migration and invasion. The PC cells with NELFE-NDRG2 double knockdown cells were used in the panels of C-G. All experiments were repeated in triplicate. ^*P<0.05, ^**P<0.01 vs. NC group. NDRG2, N-Myc downstream-regulated gene 2; NELFE, negative elongation factor E; EMT, epithelial-to-mesenchymal transition; PC, pancreatic cancer; shRNA, short hairpin RNA; NC, negative control.

Figure 5

NELFE decreased NDRG2 by interacting with its mRNA. (A) Reverse transcription-quantitative PCR results revealed that the mRNA levels of NDRG2 were increased following the knockdown of NELFE. (B) The half-life NDRG2 mRNA increased after NELFE knockdown in PC cells. (C) The decreased NELFE enhanced the luciferase activity of NDRG2 3′untranslated region. (D) RNA immunoprecipitation assays demonstration NELFE directly binding to NDRG2 mRNA. All experiments were repeated three times. ^*P<0.05, ^**P<0.01 vs. NC group. NEFLE, negative elongation factor E; NDRG2, N-Myc elongation factor E; PC, pancreatic cancer.