MicroRNA-140-5p suppresses cell proliferation and invasion in gastric cancer by targeting WNT1 in the WNT/β-catenin signaling pathway

Abstract

MicroRNAs have been suggested as potential regulators in gastric cancer (GC) development through affecting the expression of their target genes. Previous studies have demonstrated that miR-140-5p is downregulated in GC. However, the underlying functional role of miR-140-5p in GC remains largely unknown. The present study revealed that miR-140-5p expression was significantly decreased in 60 GC tissues, compared with corresponding adjacent non-tumor tissues. A lower miR-140-5p expression was significantly associated with lymph node metastasis and an advanced Tumor-Node-Metastasis stage in patients with GC. Furthermore, patients with a lower miR-140-5p expression exhibited shorter disease-free survival and overall survival times. Gain- and loss-of-function assays revealed that increased miR-140-5p expression significantly inhibited GC cell proliferation and invasion ability, as well as the Wnt/β-catenin signaling pathway by decreasing WNT1 and β-catenin expression. However, decreasing miR-140-5p expression had the opposite effects. Bioinformatics methods and dual-luciferase reporter assays revealed that WNT1 was a direct target of miR-140-5p. miR-140-5p suppressed cell proliferation and invasion by regulating WNT1 expression. Therefore, the results of the present study demonstrated that miR-140-5p may serve as a potential prognostic marker and therapeutic target in patients with GC.

Keywords: WNT1; cell invasion; cell proliferation; gastric cancer; microRNA-140-5p.

Figure 1.

Expression of miR-140-5p is significantly downregulated in GC tissues and cells. (A) The expression of miR-140-5p in 60 GC tissues and adjacent non-tumor tissues was detected using reverse transcription-quantitative polymerase chain reaction analyses. Association between miR-140-5p expression and (B) lymph node metastasis or (C) TNM stage in patients with GC. Association between miR-140-5p expression and (D) disease free survival or (E) overall survival time in patients with GC was analyzed using Kaplan-Meier survival plot analysis and the log-rank test. **P<0.01, *P<0.05. GC, gastric cancer; miR, microRNA; TNM, Tumor-Node-Metastasis.

Figure 2.

Upregulation of miR-140-5p suppresses cell proliferation and cell colony formation ability in GC. (A) The expression of miR-140-5p in the human GC MKN-45, BGC-823 and SGC-7901 cell lines, and the human immortalized normal gastric mucosa GES-1 cell line was detected using RT-qPCR analyses. (B) The expression of miR-140-5p was detected using RT-qPCR analyses following the transfection of MKN-45 cells with mimic-control or miR-140-5p mimic. (C) The expression of miR-140-5p was detected using RT-qPCR analyses following the transfection of BGC-823 cells with inhibitor-control or miR-140-5p inhibitor. (D) The results of a CCK8 assay following the transfection of MKN-45 cells with mimic-control and miR-140-5p mimic, or the transfection of BGC-823 cells with inhibitor-control and miR-140-5p inhibitor. (E) The results of cell colony formation assays following the transfection of MKN-45 cells with mimic-control and miR-140-5p mimic, or the transfection of BGC-823 cells with inhibitor-control and miR-140-5p inhibitor. Data are presented as the mean ± standard deviation of 3 independent experiments. *P<0.05. GC, gastric cancer; miR, microRNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; OD, optical density.

Figure 3.

miR-140-5p suppresses cell invasion and the Wnt/β-catenin signaling pathway in gastric cancer. The results of Transwell cell invasion assays following the transfection of (A) MKN-45 cells with mimic-control and miR-140-5p mimic, or the transfection of (B) BGC-823 cells with inhibitor-control and miR-140-5p inhibitor. The relative protein expression of WNT1 and β-catenin was detected using western blot following the transfection of (C) MKN-45 cells with mimic-control and miR-140-5p mimic, or the transfection of (D) BGC-823 cells with inhibitor-control and miR-140-5p inhibitor. Data are presented as the mean ± standard deviation of 3 independent experiments. *P<0.05. miR, microRNA.

Figure 4.

WNT1 is a direct target of miR-140-5p. (A) WNT1 was revealed to be a potential target of miR-140-5p by searching miRanda (www.microRNA.org). The WT 3′-UTR of WNT1 mRNA, which had a putative miR-140-5p binding site, and Mut 3′-UTR of WNT1 were inserted into pMIRGLO vectors. (B) miR-140-5p overexpression decreased luciferase activity of the WT 3′-UTR of the WNT1 vector, but not that of the Mut 3′-UTR of the WNT1 in MKN45 cells. The relative mRNA expression of WNT1 was detected following the transfection of (C) MKN-45 or (D) BGC-823 cells with control, miR-140-5p mimic or miR-140-5p inhibitor. Data are presented as the mean ± standard deviation of 3 independent experiments. *P<0.05. miR, microRNA; WT, wild-type; Mut, mutated-type; UTR, untranslated region.

Figure 5.

Overexpression of WNT1 reverses the effects of miR-140-5p on cell proliferation and invasion of GC. (A) The expression of WNT1 in 60 cases of GC tissues and adjacent non-tumor tissues were detected using RT-qPCR analyses. (B) The expression of WNT1 in MKN-45 cells was detected using RT-qPCR analyses when cells were transfected with empty vector or pcDNA3.1-WNT1. (C) The results of aCCK8 assay following the transfection of MKN-45 cells with mimic-control and miR-140-5p mimic or miR-140-5p mimic plus pcDNA3.1-WNT1. (D) The results of cell colony formation assays following the transfection of MKN-45 cells with mimic-control and miR-140-5p mimic or miR-140-5p mimic plus pcDNA3.1-WNT1. Data are presented as the mean ± standard deviation of 3 independent experiments. **P<0.01, *P<0.05, ^#not significant. GC, gastric cancer; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; miR, microRNA.