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. 2017 Jun 13;8(24):39280-39295.
doi: 10.18632/oncotarget.16849.

The SNAIL/miR-128 axis regulated growth, invasion, metastasis, and epithelial-to-mesenchymal transition of gastric cancer

Wei-Wei Yu  1 , Hong Jiang  1 , Cun-Tai Zhang  1 , Yang Peng  1
Affiliations

The SNAIL/miR-128 axis regulated growth, invasion, metastasis, and epithelial-to-mesenchymal transition of gastric cancer

Wei-Wei Yu et al. Oncotarget. .

Abstract

miR-128 is expressed in various tumors, but its expression and function in gastric cancer have not been defined. Thus, the goal of this study was to characterize miR-128 in gastric cancer. We found first that miR-128 is down-regulated in gastric cancer cell lines and tissues, and this dysregulation is correlated with DNA methylation and the transcription factor SNAIL. Using prediction tools, western blotting, and luciferase reporter assays, we found that Bmi-1 was the direct target of miR-128. Additionally, overexpression of miR-128 inhibited gastric cancer cell migration, invasion, and proliferation by targeting Bmi-1 in vitro and in vivo. We also documented, with receiver operating characteristic curves and Kaplan-Meier survival analysis, that miR-128 and Bmi-1 may be useful markers for diagnosing and estimating the prognosis of gastric cancer patients. As the epithelial-to-mesenchymal transition is an important mechanism associated with cancer invasion and metastasis, we inferred that miR-128 could regulate this mechanism in gastric cancer. In fact, we found that miR-128 could reverse epithelial-to-mesenchymal transition induced by Bmi-1 via the PI3K/AKT pathway. Because SNAIL also acts as a mesenchymal marker, our findings identified a novel positive feedback loop in which the transcription factor SNAIL curbs the expression of miR-128, and then down-regulated miR-128 promotes the expression of Bmi-1; finally, overexpression of Bmi-1 drives the epithelial-to-mesenchymal transition process via the PI3K/AKT pathway, and the expression of SNAIL is up-regulated.

Keywords: DNA methylation; SNAIL; gastric cancer; miR-128; transcription factor.

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Conflict of interest statement

CONFLICTS OF INTEREST

There are no conflicts of interest to declare.

Figures

Figure 1
Figure 1. miR-128 is down-regulated in gastric cancer tissues and cell lines
(A) miR-128 expression was detected in gastric cancer cell lines (SGC7901, HGC27, AGS, MKN45, and NCI-N87) and a normal gastric mucosa cell line, GES1. Data are shown as the mean ± s.d. (n = 3) in cell lines; * p < 0.05. (B) The expression level of mature miR-128 in gastric cancer (n = 135) or adjacent normal mucosa tissues (n = 135) was determined by qRT-PCR analysis. Data are shown separately in human samples; * p<0.05. (C) Mature miR-128 expression levels in metastatic (n = 83) and non-metastatic (n = 52) gastric cancers. Data are shown separately in human samples; * p < 0.05.
Figure 2
Figure 2. Methylation status of miR-128 CpG island
(A) miR-128 methylation was detected by MSP analysis in gastric cancer cell lines. (B) miR-128 methylation was detected by MSP analysis with or without 5-aza-dCyd treatment in gastric cancer cell lines. M: MSP of methylation-specific primers; U: MSP of non-methylation-specific primers. (C) miR-128 expression was examined by qRT-PCR in gastric cancer cell lines and a normal gastric mucosa cell line. Data are shown as the mean ± s.d. (n = 3) in cell lines; * p < 0.05.
Figure 3
Figure 3. miR-128 is directly repressed by SNAIL
(A) The SNAIL, TWIST, and ZEB1 expressing vector was co-transfected with various constructs containing the 3-kb fragment upstream of human miR-128-2 stem-loop into AGS cells. (B) si-SNAIL was co-transfected with miR-128 stem-loop plasmid into AGS cell lines, and luciferase activity was recorded. (C) ChIP assay analysis was performed in AGS cells transfected with a vector expressing SNAIL. Data are shown as the mean ± s.d. (n = 3) in cell lines; * p < 0.05.
Figure 4
Figure 4. miR-128 can directly target Bmi-1 expression
(A) miR-128 expression was detected in the AGS gastric cell line after transfection with miR-128 mimics or cont-miR. (B) Expression of 10 proteins was examined with western blot in the AGS gastric cell line after transfection with miR-128 mimics or cont-miR. (C) The relative luciferase activity was analyzed after the Bmi-1 reporter plasmids were co-transfected with miR-128 mimics or control mimics into AGS cell lines. (D) The relative luciferase activity was analyzed after the TGFβR1 reporter plasmids were co-transfected with miR-128 mimics or control mimics into AGS cell lines. Data are shown as the mean ± s.d. (n = 3) in cell lines; * p < 0.05, # p > 0.05.
Figure 5
Figure 5. miR-128 inhibits gastric cancer migration, invasion, and proliferation by targeting Bmi-1
(A), (B) Cell proliferation and the colony-forming ability was shown after transfection with miR-128 mimics or Cont-miR in gastric cancer cell lines AGS and MKN45. (C) The migration and invasion of AGS and MKN45 cell lines are illustrated after transfection with miR-128 mimics or Cont-miR. (D) Immunoblot analysis of Bmi-1 expression in AGS cells transfected with miR-128 mimics or cont-miR, with or without Bmi-1 restoration. (E) The tumourigenic qualities of AGS cells were assessed after transfection with miR-128 or cont-miR, with or without Bmi-1 restoration. Cell proliferation is illustrated in the left panel. Gastric cancer migration and invasion are illustrated in the right panel. Data are shown as the mean ± s.d. (n = 3) in cell lines; * p < 0.05, # p > 0.05.
Figure 6
Figure 6. miR-128 promotes an epithelial phenotype in gastric cancer
(A) Bmi-1 expression was detected with western blot in AGS cells after treatment with 3 independent siRNA sequences (si-Bmi1) or a control (si-Cont). (B) An immunoblot analysis of N-cadherin, vimentin, fibronectin, E-cadherin, and SNAIL in AGS cells transfected with si-Bmi1 or si-Cont. (C) Immunoblot analysis of pho-AKT and total-AKT in AGS cells transfected with si-Bmi1 or si-Cont. (D) Immunoblot analysis of N-cadherin, vimentin, fibronectin, E-cadherin, and SNAIL in AGS cells transfected with miR-128 mimics or cont-miR, with or without Bmi-1 restoration. Data are shown as the mean ± s.d. (n = 3) in cell lines; * p < 0.05.
Figure 7
Figure 7. Diagnostic and prognostic significance of miR-130a in gastric cancer
(A) Bmi-1 protein expression was examined with western blot in gastric cancer tissues; miR-128 expression and Bmi-1 protein expression were correlated; p < 0.05. (B) ROC curve analysis showing performance of miR-128 and Bmi-1 expression to discriminate between malignant and non-malignant tissue samples. (C) Kaplan-Meier analysis of patients' overall survival based on miR-128 and Bmi-1 expression.
Figure 8
Figure 8. miR-128 decreases tumor growth and metastasis by Bmi-1 in nude mice
(A) miR-128 expression in tumor xenografts was measured with qRT-PCR. (B) Bmi-1 expression in tumor xenografts was detected with immunohistochemical staining. (C) Images of xenograft tumors from nude mice with forced expression of miR-128 or cont-miR, with or without Bmi-1 restoration. (D) Evaluation of nuclear BrdU incorporation and TUNEL-positive (apoptotic) nuclei in xenograft tumors cells. (E) A representative IVIS imaging for metastasis in nude mice that had received tail-vein injection of miR-128 or cont-miR, with or without Bmi-1 infected AGS cells. (F) IVIS imaging for pulmonary metastasis foci and quantification for them pulmonary metastasis foci of three groups. (G) Hematoxylin and eosin-stained sections of lungs isolated from nude mice and pulmonary metastases detected in the lungs (red arrow). The data represent the means ± s.d.; * p < 0.05, # p > 0.05.
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