CPEB4 promotes growth and metastasis of gastric cancer cells via ZEB1-mediated epithelial- mesenchymal transition

Abstract

Background: Cytoplasmic polyadenylation element-binding protein 4 (CPEB4) has previously been reported to be associated with biological malignancy in various cancers. However, its function in tumor growth and metastasis in gastric cancer (GC) remains obscure. Here, we explored the functional and molecular mechanisms by which CPEB4 influences GC.

Materials and methods: The expression of CPEB4 was assessed using Western blot and immunohistochemistry in GC specimens. The roles of CPEB4 in GC cell proliferation, migration, and invasion were investigated by cell-counting kit-8 (CCK-8), colony formation, and EdU assay; wound-healing assay; and transwell assay, respectively. Quantitative real-time PCR (qRT-PCR), Western blot, and immunofluorescence staining were performed to detect the expressions of CPEB4 and epithelial-mesenchymal transition (EMT)-related markers. The function of CPEB4 on GC cell growth and metastasis was also determined in vivo through establishing subcutaneous xenograft tumor and lung metastatic mice model.

Results: The results revealed that the expression of CPEB4 was increased in GC tissues compared with matched normal tissues. High expression level of CPEB4 was significantly associated with clinical metastasis and unfavorable prognosis in patients with GC. Furthermore, CPEB4 silencing remarkably inhibited GC cells' proliferation, invasion, and metastasis in vitro and in vivo. Conversely, CPEB4 overexpression achieved the opposite effects. Mechanically, we proved that ZEB1-mediated EMT might be involved in CPEB4-facilitated GC cells' proliferation, invasion, and metastasis.

Conclusion: Our findings implied that CPEB4 expression predicted a worse prognosis in patients with GC. Besides, CPEB4 contributed to GC cells' proliferation, migration, and invasion via ZEB1-mediated EMT.

Keywords: CPEB4; epithelial-mesenchymal transition; gastric cancer.

Figure 1

Relative CPEB4 expression in GC tissues and its clinical significance. Notes: (A) Western blot analysis of CPEB4 expression in GC tumor tissues and matched normal tissues. Magnification ×200. (B) Representative IHC images of CPEB4 and ZEB1 in GC tumor tissues and adjacent normal tissues. (C) Quantitative evaluation of CPEB4 expression in tumor tissues and corresponding normal tissues in accordance with staining scores. (D) Scatterplots of the average staining scores of CPEB4 expression in patients without or with metastasis. (E) Kaplan–Meier survival curves for GC patients with CPEB4 expression. ^*P<0.05. Abbreviations: CPEB4, cytoplasmic polyadenylation element-binding protein 4; GC, gastric cancer; IHC, immunohistochemistry.

Figure 2

Effects of CPEB4 silencing or overexpression on GC cells’ migration, invasion, and proliferation in vitro. Notes: (A) Detection of relative expression of CPEB4 protein in GC cell lines and normal gastric epithelial GES-1 cells by Western blot. (B and C) Successful knockdown of CPEB4 in SGC7901 cells and CPEB4 overexpression in AGS cells were confirmed by Western blot and immunofluorescence. Magnification ×400. (D and E) The effects of CPEB4 expression on the migration and invasion ability of SGC7901 and AGS cells were determined by wound-healing assay and transwell assay. The effects of CPEB4 expression on the proliferation ability of SGC7901 and AGS cells were measured by the CCK-8 assay. Magnification ×100. (F), colony formation assay (G), and EdU assay (H). ^*P<0.05. Magnification ×200. Abbreviations: CCK-8, cell-counting kit-8; CPEB4, cytoplasmic polyadenylation element-binding protein 4; GC, gastric cancer.

Figure 3

Effects of different expression levels of CPEB4 on EMT-related markers in SGC7901 and AGS cells. Notes: Following CPEB4 shRNA or overexpression treatment, qRT-PCR (A), Western blot (B), and immunofluorescence (C) were used to determine the expressions of EMT-related markers in CPEB4-altered cells, including the epithelial marker E-cadherin, the mesenchymal markers N-cadherin and Vimentin, and transcription factors (Snail, Slug, ZEB1, SIP1, and Twist). ^*P<0.05. Magnification ×400. Abbreviations: CPEB4, cytoplasmic polyadenylation element-binding protein 4; EMT, epithelial–mesenchymal transition; qRT-PCR, quantitative real-time PCR; shRNA, short hairpin RNA.

Figure 4

CPEB4 induced GC cells’ migration, invasion, and growth via ZEB1-mediated EMT. Notes: (A) Insight into the ZEB1-dependent mechanism of CPEB4-induced GC cells’ EMT by Western blot. (B) Confirmation of the ZEB1-dependent mechanism of CPEB4-facilitating GC cells’ migration and invasion by transwell assay. Exploration of the ZEB1-dependent mechanism of CPEB4-induced GC cells growth by the CCK-8 assay. Magnification ×200. (C), EdU assay (D), and colony formation assay. Magnification ×100. (E). ^*P<0.05. Abbreviations: CCK-8, cell-counting kit-8; CPEB4, cytoplasmic polyadenylation element-binding protein 4; EMT, epithelial–mesenchymal transition; GC, gastric cancer.

Figure 5

Influences of CPEB4 silencing or overexpression on GC cells’ tumor growth in vivo. Notes: (A) Representative images of tumors formed in nude mice injected subcutaneously with GC cells. Tumor volume was measured every 5 days. At 30 days after inoculation, nude mice were sacrificed and tumors were weighed. (B) Expressions of CPEB4, E-cadherin, ZEB1, N-cadherin, and Vimentin in xenograft tumors were determined in each group by Western blot. (C) Expressions of CPEB4 and Ki-67 in xenograft tumors were detected in each group by immunohistochemistry. n=6 in each group. ^*P<0.05. Magnification ×200. Abbreviations: CPEB4, cytoplasmic polyadenylation element-binding protein 4; GC, gastric cancer.

Figure 6

Effects of CPEB4 silencing or overexpression on the metastasis of GC cells in vivo. Notes: (A) The lung metastatic nodules were counted. (B) Expressions of CPEB4, E-cadherin, ZEB1, N-cadherin, and Vimentin in lung metastatic nodules were determined in each group by Western blot. n=6 in each group. ^*P<0.05. Abbreviations: CPEB4, cytoplasmic polyadenylation element-binding protein 4; GC, gastric cancer.