MicroRNA-9 inhibits gastric cancer cell proliferation and migration by targeting neuropilin-1

Abstract

Gastric cancer (GC) is a global health problem with poor clinical outcomes. The mechanism of its development and progression remains largely unclear. The present study investigated the role of microRNA-9 (miR-9-5p) in the development and progression of GC. Overexpression of miR-9-5p led to reduced expression of neuropilin-1 (NRP-1) in GC cells. Dual-luciferase reporter assay results indicated that miR-9-5p directly targeted NRP-1. Furthermore, overexpression of miR-9-5p in GC cells increased the expression of mesenchymal markers, N-cadherin and vimentin, and decreased the expression of epithelial markers, E-cadherin and β-catenin. Overexpression of miR-9-5p inhibited GC cell proliferation, migration and invasion, and increased the sensitivity of GC cells to the anti-cancer drug cisplatin. By contrast, the opposite effects were observed in GC cells following downregulation of miR-9-5p. Taken together, the present findings suggested that miR-9-5p suppressed NRP-1 expression and inhibited GC cell proliferation and invasion. In addition, miR-9-5p overexpression attenuated GC cell resistance to anti-cancer drugs, which highlighted the potential of miR-9-5p as a target for the treatment of GC.

Keywords: gastric cancer; microRNA-9; neuropilin-1.

Figure 1.

miR-9-5p suppresses NRP-1 expression in GC cells. (A) Expression of miR-9-5p was upregulated following transfection with its mimics and downregulated by its inhibitor as determined by RT-qPCR. (B) Western blot analysis and (C) RT-qPCR demonstrated that NRP-1 expression increased in GC cells treated with miR-9-5p inhibitor, and decreased in GC cells following miR-9-5p overexpression. (D) Design of WT NRP-1 3′-UTR and Mut NRP-1 3′-UTR. Dual-luciferase assay demonstrated that miR-9-5p inhibitor increased NRP-1 3′-UTR transcriptional activities and miR-9-5p mimic had the opposite effect in (E) MKN-45 and (F) HGC-27 cells. Co-transfection with mutant NRP-1 3′-UTR inhibitor and mimic did not change the activities. *P<0.05 vs. miR-9-5p inhibitor control group; ^#P<0.05 vs. miR-9-5p-5p mimics control group. miR, microRNA; NRP-1, neuropilin-1; GC, gastric cancer; RT-qPCR, reverse transcription-quantitative PCR; UTR, untranslated region; WT, wild-type; Mut, mutant.

Figure 2.

miR-9-5p inhibits EMT in GC cells by targeting NRP-1. (A) Western blot analysis of mesenchymal and epithelial proteins. (B) Reverse transcription-quantitative PCR demonstrated that miR-9-5p inhibitor increased expression of E-cadherin and (C) β-catenin, and decreased (D) N-cadherin and (E) vimentin expression whilst overexpression of miR-9-5p produced the opposite effect. *P<0.05 vs. miR-9-5p inhibitor control group; ^#P<0.05 vs. miR-9-5p mimics control group. miR, microRNA; EMT, epithelial-mesenchymal transition; GC, gastric cancer; NRP-1, neuropilin-1.

Figure 3.

miR-9-5p reduces GC cell migration and invasion. (A) Migration assay demonstrated that downregulation of miR-9-5p increased GC cell migration whilst overexpression of miR-9-5p decreased GC cell migration. (B) Transwell assay demonstrated that miR-9-5p inhibitor increased cell invasion whilst upregulation of miR-9-5p decreased GC cell invasion. Magnification, ×40. *P<0.05 vs. miR-9-5p inhibitor control group; ^#P<0.05 vs. miR-9-5p mimics control group. miR, microRNA; GC, gastric cancer.

Figure 4.

miR-9-5p inhibits cell proliferation and drug resistance of GC cells. (A) Colony-formation assay demonstrated that GC cells transfected with miR-9-5p inhibitor increased colony-formation whilst the opposite effects were observed when GC cells overexpressed miR-9-5p. (B) Flow cytometry demonstrated that miR-9-5p inhibitor increased GC cell apoptosis, whilst miR-9-5p mimic decreased the apoptosis of GC cells following treatment with 10 µg/ml cisplatin. *P<0.05 vs. miR-9-5p inhibitor control group; ^#P<0.05 vs. miR-9-5p mimics control group. miR, microRNA; GC, gastric cancer; PI, propidium iodide.