miR-18a-5p promotes cell invasion and migration of osteosarcoma by directly targeting IRF2

Abstract

An increasing number of studies have suggested that microRNAs (miRNAs) are involved in the progress of many human cancers including osteosarcoma (OS). Especially, microRNA-18a-5p (miR-18a-5p) has been reported to associate with the occurrence, development and clinical outcomes of human cancers. Therefore, we investigated the functions of miR-18a-5p in OS. Reverse transcription-quantitative PCR (RT-qPCR) showed that miR-18a-5p was significantly upregulated in OS tissues and cell lines (MG-63 and Saos-2). The overexpression of miR-18a-5p was found to significantly promote cell migration and invasion in MG-63 cells via Transwell assay. Moreover, luciferase reporter assays indicated that interferon regulatory factor (IRF)2 was a direct target of miR-18a-5p. IRF2 was downregulated in MG-63 and Saos-2 cell lines. Furthermore, Transwell analysis showed that the knockout of IRF2 promoted cell migration and invasion in MG-63 cells. Carcinogenesis of miR-18a-5p was reversed by the overexpression of IRF2 in OS. In conclusion, miR-18a-5p promoted the invasion and migration of OS cells through inhibiting IRF2 expression. Thus, miR-18a-5p might act as a potential target for the diagnosis and treatment of OS in the future.

Keywords: interferon regulatory factor 2; invasion; miR-18a-5p; migration; osteosarcoma.

Figure 1.

miR-18a-5p expression was increased in osteosarcoma tissues and cell lines. (A) The miR-18a-5p expression was upregulated in osteosarcoma tissues. (B) The expression of miR-18a-5p was related to the tumor pathological stage of osteosarcoma. (C) The miR-18a-5p expression was increased in MG-63 and Saos-2 cells compared with normal osteosarcoma cells (hFOB1.19). **P<0.01. miR-18a-5p, microRNA-18a-5p.

Figure 2.

Overexpression of miR-18a-5p promotes cell migration and invasion in OS. (A) MG-63 cells were transfected with miR-18a-5p mimic or inhibitor, and then miR-18a-5p expression was measured via RT-qPCR. (B and C) The cell migration and invasion were detected in MG-63 cells with miR-18a-5p mimic or inhibitor. *P<0.05; **P<0.01. miR-18a-5p, microRNA-18a-5p; OS, osteosarcoma; NC, normal control without miR-18a-5p mimic or inhibitor.

Figure 3.

miR-18a-5p directly targets IRF2 in OS. (A) The binding sites of miR-18a-5p and the wild-type of IRF2. (B) Luciferase reporter assay. (C) Pearson's correlation coefficient between miR-18a-5p and IRF2 in OS tissues. (D) The mRNA expression of IRF2 was analyzed in cells transfected with miR-18a-5p mimic or inhibitor. (E) The protein expression of IRF2 was analyzed in cells transfected with miR-18a-5p mimic or inhibitor (there was a break in the gel due to the use of separate membranes). *P<0.05, **P<0.01. miR-18a-5p, microRNA-18a-5p; IRF2, interferon regulatory factor 2; OS, osteosarcoma; NC, normal control without miR-18a-5p mimic or inhibitor.

Figure 4.

Knockout of IRF2 promotes cell migration and invasion in OS. (A) IRF2 expression was decreased in MG-63 and Saos-2 cells compared with normal OS cells (hFOB1.19). (B) IRF2 expression was examined through RT-qPCR in MG-63 cells transfected with si-IRF2. (C and D) Cell migration and invasion were detected in MG-63 cells with si-IRF2. **P<0.01. IRF2, interferon regulatory factor 2; OS, osteosarcoma; si-IRF2, IRF2 siRNA; NC, normal control without si-IRF2.

Figure 5.

Overexpression of IRF2 counteracts the promoted effect of miR-18a-5p in OS. (A and B) The mRNA and protein expression of IRF2 were analyzed in cells transfected with IRF2 plasmid and miR-18a-5p mimics. (C and D) Cell migration and invasion were detected in MG-63 cells with IRF2 plasmid and miR-18a-5p mimics. **P<0.01. IRF2, interferon regulatory factor 2; miR-18a-5p, microRNA-18a-5p; OS, osteosarcoma; NC, normal control without IRF2 plasmid and miR-18a-5p mimics.