MicroRNA-124 acts as a tumor-suppressive miRNA by inhibiting the expression of Snail2 in osteosarcoma

Abstract

The aim of the present study was to investigate the clinical significance of hsa-microRNA-124-3p (miR-124) in osteosarcoma (OS), and examine its role in cell growth and invasion. Using a microRNA chip array, the expression of miR-124 was detected in samples of surgically resected OS and matched against the levels of expression in tumor-adjacent normal tissues. The levels of miR-124 were upregulated in the OS cells through the transfection of miR-124 mimics. Cell proliferation and Transwell assays were performed to determine cell proliferation and invasion; Reverse transcription-quantitative polymerase chain reaction, western blot and luciferase assays were then used to detect the expression of the target gene snail family zinc finger 2 (Snail2). The expression of miR-124 was significantly lower in the OS tissues, compared with that in the tumor-adjacent normal tissues; and the expression of miR-124 in the tumor tissues was significantly associated with tumor size. miR-124 directly repressed the expression of Snail2, and resulted in a significant inhibition of cell proliferation and invasion. In a mouse model, the overexpression of miR-124 significantly inhibited U2OS cell proliferation and invasion. Taken together, miR-124 was associated with the adverse clinical and pathological features observed in OS. It acted as a tumor suppressor to regulate the proliferation and invasion of OS cells by targeting Snail2, suggesting that miR-124 may be key in the progression of OS.

Keywords: cell invasion; microRNA-124; osteosarcoma; snail family zinc finger 2; tumor growth.

Figure 1.

Histologic assessment of adjacent normal bone tissues and OS tissues. Adjacent normal tissues: (A) Photomicrograph showing normal bone tissue and connective tissue cells consistent and neatly arranged. Arrow shows the interface between bone and connective tissue. (B) Photomicrograph showing normal cartilage osteogenesis (arrow), connective tissue and osteogenic tissue structure are normal. (C) Mature osteoblasts, connective tissue was closely arranged. arrow shows the interface between mature osteoblasts and connective tissue. OS tissues: (D) Photomicrograph showing necrotic tissue (arrow), different sizes of surrounding, darker staining and pathological mitosis. (E) Photomicrograph showing cells with deformed polykaryocytes, pathological mitosis and surrounded by malignancies into the spindle cells (arrow). (F) Photomicrograph showing tumor-producing osteoid (arrows) and pleomorphic osteoblasts. Hematoxylin and eosin staining (magnification, ×40). OS, osteosarcoma.

Figure 2.

miR-124 is downregulated in human OS. Expression profiling of miRs in human OS specimens and normal OS-adjacent tissues. Red indicates that the level of gene expression is higher than the median, and green indicates that the level is lower than the median. miR, microRNA; OS, osteosarcoma.

Figure 3.

Colony formation assay of miR-124 mimic- and miR negative control-transfected U2OS and Saos-2 cells. Colonies were stained with crystal violet in the colony formation assay for 14 days. (A) Representative micrographs of colony formation of U2OS cells. (B) Quantitative analysis of the number of U2OS colonies. (C) Quantitative analysis of the number of Saos-2 colonies. Magnification, ×200. **P<0.01, compared with cells transfected with the miR-control.

Figure 4.

Overexpression of miR-124 inhibits OS cell growth. (A) CCK-8 assay showed that miR-124 mimic U2OS cells grew more slowly, compared with cells transfected with miR-control. (B) CCK-8 assay showed that miR-124 mimic Saos-2 cells grew more slowly, compared with cells transfected with the miR-control. All data are shown as the mean ± standard deviation. **P<0.01 vs. cells transfected with the miR-control. miR, microRNA; OS, osterosarcoma; CCK-8, Cell Counting Kit-8.

Figure 5.

miR-124 significantly suppresses U2OS cell and Saos-2 cell invasion. (A) Representative micrographs of transmembrane invasion of U2OS cells on the outer membrane (magnification, ×200). Quantitative analysis of average numbers of invading (B) U2OS cells and (C) Saos-2 cells from three randomly selected visual fields. U2OS cells and Saos-2 cells were transfected with 100 nM of either miR-124 mimics or miR-control for 12 h, and then 5×10⁴ cells/500 µl were plated in the top chamber. Low-serum medium (0.5% FBS) was added to the lower chamber. Following incubation for 24 h at 37°C, the cells on the lower surface were fixed with 10% paraformaldehyde for 30 min, stained with crystal violet staining solution, and images were captured with a digital camera. miR, microRNA; **P<0.01, compared with cells transfected with the miR-control.

Figure 6.

Silencing of miR-124 inhibits osteosarcoma tumor formation in vivo. (A) Tumor formation was evidenced by tumor growth rates in three groups; tumors in the miR-124 group were smaller than those in the control group. (B) Analysis of tumor weights. **P<0.01, compared with cells transfected with the miR-control; miR, microRNA; NC, negative control (empty vector).

Figure 7.

Prediction and validation of Snail2 as a target of miR-124. (A) Diagram illustrating the hypothetical duplexes formed by interactions between the binding sites in human and mouse miR-124 (top) and the Snail2 3′UTR (bottom). The seed recognition sites and mutation of the binding site are shown; all nucleotides in these regions were highly conserved across species, including humans and mice. (B) Analysis of the reporter activity in U2OS cells and Saos-2 was performed to confirm whether miR-124 directly targeted the predicted three binding sites in the Snail2 3′UTR and negatively regulates the expression of Snail2. (C) Reverse transcription-quantitative polymerase chain reaction analysis of the expression levels of Snail2 in U2OS cells treated with control mimic or miR-124 mimic. (D) Western blot analysis of the effect of miR-124 on the protein expression of Snail2 in U2OS cells. miR, microRNA; UTR, untranslated region; wt, wild-type; *P<0.05, **P<0.01, compared with cells transfected with the miR-control.