MIR-519d suppresses the gastric cancer epithelial-mesenchymal transition via Twist1 and inhibits Wnt/β-catenin signaling pathway

Abstract

MicroRNAs (miRNAs) deregulation is frequent in human gastric cancer (GC). MiR-519d has been reported to function as tumor suppressor microRNA in some tumors. However, the role of miR-519d in GC progression remains unclear. In the study, we demonstrated that the expression of miR-519d was down-regulated in gastric cancer tissues and cell lines, and lower miR-519d expression was associated with distant metastasis, lymph node metastasis and clinical stage for patients with GC. Univariate and multivariate Cox analysis showed that lower miR-519d expression was positively associated with shorter disease-free survival (DFS) and the over survival (OS) time for GC patients and was an independent predictor. Kaplan-Meier curve and log-rank test also demonstrated that lower miR-519d had a poor shorter DFS and OS for GC patients. Function analysis showed that the inhibition of miR-519d expression was able to promote the cell proliferation, migration and invasion and over-expression of miR-519d in GC cells had inhibited effects. Moreover, we demonstrated that over-expression of miR-519d significantly inhibited the process of epithelial mesenchymal transition (EMT) in GC cells and miR-519d can directly target at 3'-untranslation region of Twist1 and regulate its expression. We also demonstrated that miR-519d could suppress the Wnt/β-catenin signaling pathway in GC cells. In vivo, we showed that miR-519d inhibited the tumor growth. Thus, our results suggested that miR-519d functioned as a tumor suppressor in GC and could be a promising therapeutic target for GC.

Keywords: Gastric cancer; Twist1; cell invasion; epithelial mesenchymal transition; miR-519d.

Figure 1

MiR-519d was down-regulated in gastric cancer tissue samples and cell lines. A. QRT-PCR analysis of miR-519d expression in human GC tissue samples and their matched normal GC tissues from 112 GC patients. B. Correlation between miR-519d expression and distant metastasis of GC. C. Correlation between miR-519d expression and lymph node metastasis of GC. D. Correlation between miR-519d expression and TNM stage of GC. E, F. Kaplan-Meier survival curve and log-rank test were performed to analyze the correlation between miR-519d expression and DFS or OS. **: P<0.05.

Figure 2

MiR-519d inhibited the cell proliferation in GC cells. A. QRT-PCR analysis of miR-519d expression in four human GC cell lines MKN45, HGC27, BGC-823, and SGC-7901 and one normal cell line GES-1. B. QRT-PCR analysis of miR-519d expression by transfecting miR-519d plasmid into MKN45 cells. C. QRT-PCR analysis of miR-519d expression by transfecting antagomiR-519d into SGC-7901 cells. D. CCK8 cell proliferation assays were used to analyze the cell proliferation ability by transfecting miR-519d plasmid or negative control into MKN45 cells. E. CCK8 cell proliferation assays were used to analyze the cell proliferation ability by transfecting antagomiR-519d or negative control into SGC-7901 cells. Data are means ± SD of three independent experiments. **: P<0.05.

Figure 3

MiR-519d inhibited the cell migration and invasion in GC cells. A, B. Transwell cell migration assays and cell number analysis were used to analyze the cell migration ability by transfecting miR-519d plasmid or negative control into MKN45 cells. C, D. Transwell cell migration assays and cell number analysis were used to analyze the cell migration ability by transfecting antagomiR-519d or negative control into SGC-7901 cells. E, F. Transwell cell invasion assays and cell number analysis were used to analyze the cell invasion ability by transfecting miR-519d plasmid or negative control into MKN45 cells. G, H. Transwell cell invasion assays and cell number analysis were used to analyze the cell invasion ability by transfecting antagomiR-519d or negative control into SGC-7901 cells. Data are means ± SD of three independent experiments. **: P<0.05.

Figure 4

MiR-519d inhibited the cell EMT by targeting Twist1 in GC cells. A. Western-blot analysis was performed to analyze the protein expression of Twist1, E-cadherin, N-cadherin and Vimentin by transfecting miR-519d plasmid or negative control into MKN45 cells, the β-actin was used the internal control. B. western-blotting analysis was performed to analyze the protein expression of Twist1, E-cadherin, N-cadherin and Vimentin by transfecting antagomiR-519d or negative control into SGC-7901 cells, the β-actin was used the internal control. C. Bioinformatics analysis of between miR-519d and Twist1 recognition sequences by miRanda (http://mircorna.org) revealed the miR-519d had a binding site with Twist1. D, E. miR-519d mimic decreased the luciferase activities in Twist1-WT + miR-519d mimic group, but did not affect luciferase activity by transfecting miR-519d mimic + Twist1-MUT group into MKN45 or SGC-7901 cells. F, G. The Twist1 expression was determined by qRT-PCR analysis by transfecing miR-519d plasmid or antagomiR-519d into MKN45 cells. Data are means ± SD of three independent experiments. **: P<0.05.

Figure 5

MiR-519d suppresses the Wnt/β-catenin signaling pathway in GC cells. A. Western-blot analysis was performed to analyze the protein expression of β-catenin, p-GSK-3β, GSK-3β, NKD1 by transfecting miR-519d plasmid or negative control into MKN45 cells, the β-actin was used the internal control. B. Western-blot analysis was performed to analyze the protein expression of β-catenin, p-GSK-3β, GSK-3β, NKD1 by transfecting antagomiR-519d or negative control into SGC-7901 cells, the β-actin was used the internal control. C. The tumor was showed in pre-miR-519d plasmid or negative control group. D. The tumor size was showed in pre-miR-519d plasmid or negative control group. E. Western-blot analysis was performed to analyze the protein expression of Twist1 in pre-miR-519d plasmid or negative control group. Data are means ± SD of three independent experiments. **: P<0.05.