The Downregulation of MiR-182 Is Associated with the Growth and Invasion of Osteosarcoma Cells through the Regulation of TIAM1 Expression

Abstract

Background: Osteosarcoma is the most common primary bone malignancy in children and young adults. Increasing results suggest that discovery of microRNAs (miRNAs) might provide a novel therapeutical target for osteosarcoma.

Methods: MiR-182 expression level in osteosarcoma cell lines and tissues were assayed by qRT-PCR. MiRNA mimics or inhibitor were transfected for up-regulation or down-regulation of miR-182 expression. Cell function was assayed by CCK8, migration assay and invasion assay. The target genes of miR-182 were predicated by bioinformatics algorithm (TargetScan Human).

Results: MiR-182 was down-regulated in osteosarcoma tissues and cell lines. Overexpression of miR-182 inhibited tumor growth, migration and invasion. Subsequent investigation revealed that TIAM1 was a direct and functional target of miR-182 in osteosarcoma cells. Overexpression of miR-182 impaired TIAM1-induced inhibition of proliferation and invasion in osteosarcoma cells.

Conclusions: Down-expression of miR-182 in osteosarcoma promoted tumor growth, migration and invasion by targeting TIAM1. MiR-182 might act as a tumor suppressor gene whose down-regulation contributes to the progression and metastasis of osteosarcoma, providing a potential therapy target for osteosarcoma patients.

Fig 1. miR-182 was down-regulated in osteosarcoma cell lines and tissues.

(A) The expression levels of miR-182 were measured by qRT-PCR in osteosarcoma cell lines (U2OS, MG-63, SOSP-9607, and SAOS-2) and human normal osteoblastic cell line (hFOB). The expression of miR-182 was down-regulated in osteosarcoma cell lines. (B) Relative expression of miR-182 in 40 primary osteosarcoma tissues compared with their pair-matched nontumor tissues. Data are shown as log2 of relative ratio change of osteosarcoma tissues relative to normal tissues. (C) The expression of miR-182 in the human osteosarcoma tissues was lower than that in non-tumor adjacent tissues. (D) Loss of miR-182 levels in patients with osteosarcoma was associated with associated with considerably shortened disease-free survival. All data were normalized by U6 snRNA. ***p<0.001.

Fig 2. Overexpression of miR-182 inhibited tumor growth.

(A) The miR-182 expression level of the indicated cells was measured by qRT-PCR. The expression of miR-182 was normalized to U6 snRNA. (B) The expression level of miR-182 in the indicated cells was measured by qRT-PCR. The expression of miR-182 was normalized to U6 snRNA. (C) Cell proliferation of the indicated cells was analyzed using CCK-8 assay. (D) Inhibition of miR-182 promote the osteosarcoma cell proliferation. *p<0.05, ** p<0.01, and ***p<0.001.

Fig 3. Overexpression of miR-182 inhibited tumor invasion.

(A) Wound-healing assay was employed to evaluate the migratory ability of MG-63 cells after treatment with miR-182 mimics, inhibitors or scramble or no transfection. Relative ratio of wound closure is shown on the right. (B) Invasion analysis were employed to evaluate the invasive ability of the MG-63 cells after treatment with miR-182 mimics, inhibitors or scramble or no transfection; the relative ratio of invasive cells per field is shown on the right, *p<0.05, ** p<0.01, and ***p<0.001.

Fig 4. miR-182 directly targets the TIAM1 in osteosarcoma cells.

(A) The potential miR-182 binding site at the 3'-UTR of TIAM1 mRNA was computationally predicted by Targetscan. (B) Cells were transfected with wild type 3'-UTR reporter or mutant constructs together with 20 nM of miR-182 mimics or controls. Luciferase activity was normalized by the ratio of firefly and Renilla luciferase signals. (C) Relative expression of TIAM1 in the MG-63 cells transfected with miR-182 mimics or negative control or inhibitor. The expression of TIAM1 was normalized to GAPDH. (D) Western blot analysis of TIMA1 expression in MG-63 cells transfected with miR-182 mimics or negative control or untreat group. GAPDH was also detected as a loading control. (E) Western blot analysis of TIAM1 expression in MG-63 cells transfected with miR-182 inhibitor or negative control or untreat group. GAPDH was also detected as a loading control. ***p<0.001.

Fig 5. Restoration of miR-182 inhibits TIAM1-inducing osteosarcoma cell proliferation and invasion.

(A) qRT-PCR analysis of TIAM1 expression in osteosarcoma cell lines (U2OS, MG-63, SOSP-9607, and SAOS-2) and human normal osteoblastic cell line (hFOB). The mRNA expression of TIAM1 was up-regulated in osteosarcoma cell lines. The expression of TIAM1 was normalized to GAPDH. (B) Western blot analysis of TIAM1 expression in osteosarcoma cell lines (U2OS, MG-63, SOSP-9607, and SAOS-2) and human normal osteoblastic cell line (hFOB). The protein expression of TIAM1 was up-regulated in osteosarcoma cell lines. GAPDH was also detected as a loading control. (C) The effects of pCDNA-TIAM1 on the expression of TIAM1 was detected by Western blotting. GAPDH was also detected as a loading control. (B) The cell growth in MG-63 co-transfected with either miR-182 mimic or scramble and 2.0 μg pCDNA-TIAM1 or pCDNA empty vector using CCK-8 proliferation assay. (C) The cell invasive in MG-63 co-transfected with either miR-182 mimic or scramble and 2.0 μg pCDNA-TIAM1 or pCDNA empty vector using invasion assay. *p<0.05, ** p<0.01, and ***p<0.001. Figs 5B and 5D are excluded from this article's CC-BY license. See the accompanying retraction notice for more information.