MicroRNA-379 inhibits the proliferation, migration and invasion of human osteosarcoma cells by targetting EIF4G2

Abstract

Osteosarcoma (OS) is an aggressive malignant mesenchymal neoplasm amongst adolescents. The aim of the present study was to explore the various modes of action that miR-379 has on the proliferation, migration, and invasion of human OS cells. miR-379 achieves this by targetting eukaryotic initiation factor 4GII (EIF4G2). Human OS cell lines U2OS and MG-63 were selected and assigned into blank, miR-379 mimics, miR-379 mimic negative control (NC), miR-379 inhibitors, miR-379 inhibitor NC, EIF4G2 shRNA, control shRNA, and miR-379 inhibitor + EIF4G2 shRNA group. The miR-379 expression and EIF4G2 mRNA expression were detected utilising quantitative real-time PCR (qRT-PCR) and the EIF4G2 protein expression using Western blotting. MTT assay, scratch test, Transwell assay, and flow cytometry were performed to determine the proliferation, migration, invasion, and cell cycle, respectively. In comparison with the miR-379 mimic NC group, the miR-379 mimics group had decreased EIF4G2 expression; the miR-379 inhibitors group indicated an increased EIF4G2 expression. Compared with the control shRNA group, the EIF4G2 expression was lower in the EIF4G2 shRNA group and the miR-379 expression was dropped in the miR-379 inhibitor + EIF4G2 shRNA group. The proliferation, migration, and invasion abilities of OS cells were reduced in the miR-379 mimics and EIF4G2 shRNA groups. The percentage of OS cells at the G₀/G₁ stage was increased, and the percentage at the S-stage was decreased in the miR-379 mimics and EIF4G2 shRNA groups. miR-379 may inhibit the proliferation, migration and invasion of OS cells through the down-regulation of EIF4G2.

Keywords: EIF4G2; Invasion; MicroRNA-379; Migration; Osteosarcoma; Proliferation.

Figure 1. miR-379 inhibited EIF4G2 expression in U2OS and MG-63 cell lines.

(A) miR-379 inhibited EIF4G2 expression in U2OS cells; (B) miR-379 inhibited EIF4G2 expression in MG-63 cells; (C) the protein expression of EIF4G2 in U2OS cells in each group; (D) values of the protein expression of EIF4G2 in U2OS cells in each group; (E) the protein expression of EIF4G2in MG-63 cells in each group; (F) values of the protein expression of EIF4G2 in MG-63 cells in each group; n=3; *, P<0.05 compared with the miR-379 mimic NC group; #, P<0.05 compared with the miR-379 inhibitor NC group; @, P<0.05 compared with the control shRNA group.

Figure 2. Identification of the targetting relationship between miR-379 and EIF4G2 by bioinformatics analysis and dual luciferase reporter gene assay.

(A) miR-379 was predicted to target EIF4G2 by bioinformatics software; (B) dual luciferase reporter analysis verified the targetting relationship between miR-379 and EIF4G2; n=3; *, P<0.05 compared with the miR-379 mimic NC group.

Figure 3. The proliferation of U2OS and MG-63 cell lines detected by MTT assay.

(A) The proliferation of U2OS cells in each group; (B) the proliferation of MG-63 cells in each group; n=3; #, P<0.05 compared with the miR-379 inhibitor NC group; *, P<0.05 compared with the miR-379 mimic NC group; @, P<0.05 compared with the control shRNA group.

Figure 4. The migration of U2OS and MG-63 cell lines detected by the scratch test.

(A) The migration distance of the U2OS cells in each group; (B) observation of the migration ability of the U2OS cells under a light microscope; (C) the migration distance of the MG-63 cells in each group; (D) observation of the migration ability of the MG-63 cells under a light microscope; n=3; #, P<0.05 compared with the miR-379 inhibitor NC group; *, P<0.05 compared with the miR-379 mimic NC group; @, P<0.05 compared with the control shRNA group.

Figure 5. The migration and invasion abilities of U2OS and MG-63 cell lines detected by the Transwell assay

(A) The number of invasive U2OS cells in each group; (B) images of the invasion of U2OS cells under a light microscope; (C) the number of invasive MG-63 cells in each group; (D) images of the invasion of the MG-63 cells under a light microscope; (E) the number of migrating U2OS cells in each group; (F) images of the migration of U2OS cells under a light microscope; (G) the number of migrating MG-63 cells in each group; (H) images of the migration of the MG-63 cells under a light microscope; n=3; #, P<0.05 compared with the miR-379 inhibitor NC group; *, P<0.05 compared with the miR-379 mimic NC group; @, P<0.05 compared with the control shRNA group.

Figure 6. Cell cycles of U2OS and MG-63 cell lines detected by flow cytometry.

(A) The cell cycle of U2OS cells in each group; (B) the cell cycle of MG-63 cells in each group; n=3; #, P<0.05 compared with the miR-379 inhibitor NC group; *, P<0.05 compared with the miR-379 mimic NC group; @, P<0.05 compared with the control shRNA group.