miR-4295 promotes cell proliferation, migration and invasion of osteosarcoma through targeting interferon regulatory factor 1

Abstract

Osteosarcoma (OS) is the most common form of primary malignant bone tumor. Despite encouraging progress in the treatment of OS, the survival rate for patients with OS has remained unchanged over the past 40 years. It has been established that miRNA plays a crucial regulatory role in the progression and development of OS. To explore the potential association of miRNAs with OS, bioinformatics techniques were used to screen for differentially expressed miRNA genes in OS in the Gene Expression Omnibus database. In the GSE70367 database, it was revealed that miR-4295 expression was abnormally elevated in the expression of OS cells. To characterize the potential function of miR-4295 in OS, the expression levels of miR-4295 in 30 samples of OS and adjacent normal tissues was examined. The results revealed that the expression of miR-4295 was significantly increased in OS tissues compared with the paired normal tissues. Moreover, the expression levels of miR-4295 in OS cell lines (MG-63 and Saos-2) were significantly higher compared with those in the normal human mesenchymal stem cells. In addition, miR-4295 was associated with OS cell proliferation, migration and invasion. Furthermore, it was demonstrated that the expression of interferon regulatory factor (IRF)1, a tumor suppressor, was regulated by miR-4295 directly in OS cells. Taken together, the present results revealed that miR-4295 may act as a tumor activator by targeting IRF1 during the progression of OS. Investigating miR-4295 may provide novel insight into the mechanisms of OS metastasis, and inhibition and targeting miR-4295 may be a novel therapeutic strategy for the treatment of OS.

Keywords: interferon regulatory factor 1; invasion; miR-4295; migration; osteosarcoma; proliferation.

Figure 1.

Expression levels of miR-4295 mRNA are significantly increased in OS tissues and cells. (A) Expression levels of miR-4295 in 30 samples of OS and adjacent normal tissues analyzed using reverse transcription PCR. Statistical analysis was evaluated using a paired Student's t-test. (B) Expression levels of miR-4295 in MG-63 and Saos-2 cell lines. Statistical analysis was evaluated using one-way analysis of variance and Tukey's post hoc test. (C) Expression levels of miR-4295 in the MG-63 cell line after transfection of miR-4295 mimics. Statistical analysis was evaluated using two-sided independent Student's t-test. **P<0.01, ***P<0.001. miR, microRNA; OS, osteosarcoma.

Figure 2.

Effects of miR-4295 on the proliferation, migration and invasion of OS cells. (A and B) Cell Counting Kit-8 assay of MG-63 and Saos-2 cells after transfection with miR-4295 mimics/mimics-NC and miR-4295 inhibitor/inhibitor-NC. Statistical analysis was evaluated using two-sided independent Student's t-tests. (C and D) Migration and invasion of MG-63 and Saos-2 cells after transfection with miR-4295 mimics/mimics-NC and miR-4295 inhibitor/inhibitor-NC. (E and F) Three fields of the indicated cells were evaluated. Means + SD are shown from three independent experiments performed in triplicate Statistical analysis was performed using two-sided independent Student's t-tests. (G and H) The wound-healing assay of MG-63 and Saos-2 cells after transfected with miR-4295 mimics/mimics-NC, miR-4295 inhibitor/inhibitor-NC, respectively. Scale bar, 200 µm. *P<0.05, **P<0.01, ***P<0.001. miR, microRNA; OS, osteosarcoma; NC, negative control.

Figure 3.

miR-4295 targets the IRF1 gene directly. (A and B) Expression levels of IRF1 after upregulation or knockdown of miR-4295 in MG-63 and Saos-2 cells. (C and D) Luciferase activity of the PMIRGLO-IRF1 reporter in MG-63 and Saos-2 cells was identified after co-transfection of the IRF1 3′UTR plasmid with miR-4295 mimics/mimics-NC or miR-4295 inhibitor/inhibitor-NC. Statistical analysis was evaluated using independent Student's t test. *P<0.05, **P<0.01, ***P<0.001. IRF1, interferon regulatory factor 1; miR, microRNA; NC, negative control; UTR, untranslated region. Scale bar, 200 µm.

Figure 4.

miR-4295 promotes Saos-2 cell migration and invasion by targeting the IRF1 gene. (A) Correlation of expression levels of miR-4295 and IRF1 in 30 samples of OS tissues analyzed using reverse transcription PCR. Statistical analysis was evaluated with the Pearson's correlation test. (B) Expression levels of IRF1 after transfection with LV–IRF1 and LV-NC lentivirus in Saos-2 cells transfected with mimics-NC or mir-4295 mimics. Statistical analysis was analyzed using one-way analysis of variance and Tukey's post hoc test. (C) Cell Counting Kit-8 assay of Saos-2 cells after transfection with LV–IRF1 and LV-NC lentivirus. Statistical analysis was analyzed using one-way analysis of variance and Tukey's post hoc test. (D) Migration and invasion of Saos-2 cells after transfection with LV–IRF1 and LV-NC lentivirus. (E) Three fields of the indicated cells were evaluated. Means + SD are shown from three independent experiments performed in duplicates. Statistical analysis was evaluated using one-way analysis of variance and Tukey's post hoc test. *P<0.05, **P<0.01. miR, microRNA; IRF1, interferon regulatory factor 1; OS, osteosarcoma; NC, negative control; ns, not significant. Scale bar, 200 µm.