miR-144 suppresses cell proliferation and invasion in gastric cancer through downregulation of activating enhancer-binding protein 4

Abstract

Gastric cancer (GC) is the most common malignant disease and its incidence rate is increasing rapidly worldwide. The molecular mechanisms underlying GC tumorigenesis require further investigation. The expression and physiological roles of microRNA-144 (miR-144) have been investigated in numerous types of tumor. However, its biological function in GC remains largely unknown. The reverse transcription- quantitative polymerase chain reaction was used to determine the expression of miR-144 in GC cells and normal gastric epithelial cells. An miR-144 mimic was transfected into HGC-27 cells. In addition, bioinformatics analysis was performed to identify the potential targets of miR-144. Protein expression, luciferase and rescue assays were used to confirm the target of miR-144. It was identified that the expression of miR-144 was significantly downregulated in GC cells compared with in normal gastric epithelial cells. Furthermore, overexpression of miR-144 suppressed HGC-27 cell proliferation, migration and invasion. Additionally, bioinformatics analysis suggested that the activating enhancer-binding protein 4 (AP4) is a target gene of miR-144. In addition, it was determined that miR-144 suppresses the expression of AP4 by binding directly to its 3'-untranslated regions. Furthermore, restoration of AP4 partially attenuated miR-144-induced inhibition of cell proliferation, migration and invasion. Therefore, the results of the present study suggest that miR-144 serves an important role in GC progression.

Keywords: activating enhancer-binding protein 4; gastric cancer; invasion; microRNA-144; proliferation.

Figure 1.

Expression levels of miR-144 in gastric cancer cells. (A) miR-144 expression was determined by RT-qPCR in gastric cancer cell lines (HGC-27, SGC-7901, MKN-45, AGS and BGC-823) and a normal gastric epithelial cell line (GES-1). ***P<0.001 vs. GES-1. (B) RT-qPCR was performed to confirm the effect of transfection with miR-144 mimic. ***P<0.001 vs. miR-NC. Data are from three independent experiments and are presented as the mean ± standard deviation. miR, microRNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; NC, negative control.

Figure 2.

miR-144 suppresses the proliferation, migration and invasion of gastric cancer cells. (A) An MTT assay was performed to examine proliferation. (B) Quantitative analysis of the percentage of apoptotic cells. (C) Representative histograms of apoptosis analysis. In each plot, viable cells are in the Q4 quadrant, early apoptotic cells are in the Q3 quadrant, late apoptotic cells are in the Q2 quadrant and necrotic cells are in the Q1 quadrant. (D) Migration assay of HGC-27 cells transfected with miR-144 or miR-NC. (E) Invasion assay of HGC-27 cells transfected with miR-144 or miR-NC. Data are from three independent experiments and are presented as the mean ± standard deviation *P<0.05, **P<0.01, ***P<0.001 vs. miR-NC. miR, microRNA; NC, negative control; OD, optical density; PI, propidium iodide; FITC, fluorescein isothiocyanate; Q, quadrant.

Figure 3.

AP4 is a direct target of miR-144. (A) WT AP4 3′-UTR and Mut AP4 3′-UTR sequences. (B) The protein expression level of AP4 in HGC-27 cells transfected with miR-NC, miR-144 or anti-miR-144 was determined by western blot analysis. (C) The luciferase activity of the WT and Mut AP4 3′-UTR co-transfected with miR-144 or miR-NC was determined. Data are from three independent experiments and are presented as the mean ± standard deviation **P<0.01 vs. miR-NC. WT, wild-type; AP4, activating enhancer-binding protein 4; 3′-UTR; 3′-untranslated region; miR, microRNA; NC, negative control.

Figure 4.

Overexpression of AP4 partially reverses the tumor-suppressive effects of miR-144. (A) HGC-27 cells were transfected with miR-144 with/without AP4 overexpression plasmid. (B) MTT, (C) migration (D) and invasion assays were performed to determine the proliferation, migration and invasion of HGC-27 cells transfected with miR-144 with/without AP4. Data are from three independent experiments and are presented as the mean ± standard deviation *P<0.05, **P<0.01 vs. miR-NC; ^#P<0.05 vs. miR-144-transfected cells. AP4, activating enhancer-binding protein 4; miR, microRNA; OD, optical density; NC, negative control.