Upregulation of microRNA-520a-3p inhibits the proliferation, migration and invasion via spindle and kinetochore associated 2 in gastric cancer

Abstract

MicroRNAs (miR) serve important roles in the development and progression of tumors by targeting different genes. miR-520a-3p reported in lung and breast cancers as a tumor suppressor gene. However, the expression and functional significance of miR-520a-3p is not completely understood in gastric cancer (GC). In the present study, it was demonstrated that the expression levels of miR-520a-3p were significantly downregulated in GC tissues and cells using RT-qPCR. In addition, downregulated expression of miR-520a-3p was associated with the clinical stage of the tumor and invasion in patients with GC. Furthermore, overexpression of miR-520a-3p significantly inhibited cell proliferation, invasion and migration in SGC-7901 and MGC-803 GC cell lines using proliferation, wound healing and cell invasion assays. Spindle and kinetochore associated 2 (SKA2) was upregulated in GC cells using western blot analysis and a target gene of miR-520a-3p; miR-520a-3p mimics significantly reduced SKA2 expression. In addition, upregulation of SKA2 protein expression SKA2 reversed the miR-520a-3p-mediated inhibition of SGC-7901 cell proliferation, migration and invasion. In conclusion, miR-520a-3p functioned as a tumor suppressor gene by targeting SKA2 in GC cell lines, and may serve as a novel prognostic and potential therapeutic marker.

Keywords: gastric cancer; invasion; microRNA-520a-3p; migration; proliferation; spindle and kinetochore-associated protein 2.

Figure 1.

miR-520a-3p expression is decreased in GC tissues and cell lines. (A) miR-520a-3p expression was detected via reverse transcription-quantitative PCR analysis in 80 GC tissues compared with adjacent non-tumor tissues. **P<0.01 vs. adjacent non-tumor tissues (B) miR-520a-3p expression was analyzed in three GC cell lines (SGC-7901, BGC-823, and MGC-803) compared with in a normal gastric epithelial cell line (GES-1). The levels of miR-520a-3p expression were normalized to U6. *P<0.05, **P<0.01 vs. GES-1. miR, microRNA; GC, gastric cancer.

Figure 2.

Overexpression of miR-520a-3p inhibits the proliferation of GC cells. (A) Following transfection with miR-520a-3p mimics, the mRNA expression levels of miR-520a-3p were significantly increased in SGC-7901 and MGC-803 cells compared with the respective MOCK and NC groups. ***P<0.001 vs. MOCK and NC. (B) GC cell viability in SGC-7901 and MGC-803 cells was examined by cell proliferation assay following the overexpression of miR-520a-3p. Transfection of the mimics significantly reduced proliferation. *P<0.05, **P<0.01 vs. MOCK and NC. (C) Western blot of the Ki-67 expression levels normalized to GAPDH. ***P<0.001 vs. MOCK and NC. miR, microRNA; GC, gastric cancer; MOCK, mock transfected cells; NC, negative control; OD, optical density.

Figure 3.

Overexpression of miR-520a-3p inhibits the migratory and invasive capabilities of GC cells. (A) Migratory distance and (B) number of invasive cells in the miR-520a-3p mimics group were significantly decreased compared with the MOCK and NC groups in the wound healing and Transwell invasion assays. *P<0.05 vs. MOCK and NC. (C) Protein expression levels of MMP-2 and MMP-9 were significantly lower in the miR-520a-3p mimics group compared with the MOCK and NC groups. ***P<0.001 vs. MOCK and NC. Magnification, ×100. Scale bar, 100 µm. miR, microRNA; GC, gastric cancer; MOCK, mock transfected cells; NC, negative control; MMP, matrix metalloproteinase.

Figure 4.

SKA2 is a direct target gene of miR-520a-3p in GC cells. (A) Sequence alignment of miR-520a-3p with 3′UTR of SKA2 predicted by TargetScan and the Mut 3′UTR miR-520a-3p binding sequence. (B) Luciferase activity of SGC-7901 cells was examined via a luciferase reporter assay. **P<0.01 vs. mutant. miR-520a-3p decreased SKA2 expression at both the mRNA (C) and protein (D) levels compared to MOCK in SGC-7901 cells. **P<0.01, ***P<0.001 vs. MOCK group. (E) SKA2 expression was analyzed in three GC cell lines (SGC-7901, BGC-823 and MGC-803) and a normal gastric epithelial cell line (GES-1). ***P<0.001 vs. GES-1. miR, microRNA; GC, gastric cancer; MOCK, mock transfected cells; NC, negative control; WT, wild-type; SKA2, spindle kinetochore-associated protein 2; UTR, untranslated region.

Figure 5.

SKA2 regulates miR-520a-3p-mediated inhibition of SGC-7901 cells proliferation, migration and invasion. (A) After cells were transfected with a SKA2 cDNA plasmid, the expression of SKA2 was notably increased in SGC-790 cells. Following co-transfection of miR-520a-3p mimics and SKA2 cDNA into SGC-7901 cells, (B) expression of SKA2 and (C) cell viability were examined by western blotting and cell proliferation assay, respectively. (D) Migration distance and (E) number of invasive cells in GC cells following treatment with miR-520a-3p mimics and SKA2 cDNA were detected by wound healing and Transwell assays, respectively. **P<0.01, ***P<0.001 vs. the MOCK group. ×100 magnification. Scale bar, 100 µm. SKA2, spindle kinetochore-associated protein 2; miR, microRNA; GC, gastric cancer; MOCK, mock transfected cells; NC, negative control; OD, optical density.