MicroRNA-217 regulates WASF3 expression and suppresses tumor growth and metastasis in osteosarcoma

Abstract

Osteosarcoma is the most common type of primary tumor of bone which mainly affects adolescents and young adults. Osteosarcoma causes large number of deaths because of its complex pathogenesis and resistance to conventional treatment. MicroRNAs are a class of small noncoding RNAs that function as critical gene regulators through targeting mRNAs, causing translational repression or degradation. In this study, we showed that miR-217 was down-regulated in osteosarcoma cell lines and tissues in comparison to that in normal bone cells or tissues. Meanwhile, the lower level of miR-217 was associated with metastasis in clinical osteosarcoma patients. Furthermore, we found that overexpession of miR-217 markedly suppressed cell proliferation, migration, and invasion of osteosarcoma cells. Conversely, the inhibition of miR-217 expression significantly accelerated the cell proliferation, migration, and invasion. Moreover, we identified WASF3 as a novel functional downstream target of miR-217. The ectopic expression of WASF3 can partially reverse the inhibition of cell proliferation and invasion caused by miR-217. Take together, our results demonstrate that miR-217 functions as a tumor-suppressive miRNA and inhibits the osteosarcoma tumorigenesis through targeting WASF3.

Figure 1. miR-217 is downregulated in human osteosarcoma cell lines and tissues.

(A) Relative expression of miR-217 in four human osteosarcoma cell lines (MG-63, U2OS, SOSP-9607, and SAOS-2) and one normal bone cell line (hFOB) was determined by qRT-PCR. Quantification of miR-217 was measured by qRT-PCR with specific primers for miR-217 and snRNA U6. (B) Relative expression of miR-217 in 60 primary osteosarcoma tissues compared with their pair-matched nontumor tissues. Data areshown as log10 of relative ratio change of osteosarcoma tissues relative tonormal tissues. (C) Relative miR-217expressionlevels in osteosarcoma tissues and adjacent normal regions; (D)The expression of miR-217 in the osteosarcoma tissues from the patients with metastases was lower than that in non-metastases tissues. All data uses t test and is shown as mean ±SEM.

Figure 2. Overexpression of miR-217 inhibits the osteosarcoma cell line MG-63 proliferation, migration, and invasion.

(A) Expression levels of miR-217 were examined by qRT-PCR after transfection of 20 nmol/L of miR-217 mimics, inhibitors or sramble or no transfection in the cell line MG-63. (B) The cells treated with miR-217 mimics, inhibitors or sramble or no transfection were measured by CCK8 assay at different time periods. (C) Wound healing assays of MG-63cells after treatment with miRNA mimics, inhibitors or scramble or no transfection; the relative ratio of wound closure per field is shown. (D) Transwell analysis of MG-63 cells after treatment withmiRNA mimics, inhibitors or scramble or no transfection; the invasive cells per field is shown below, All data uses t test and is shown as mean ±SEM. *p<0.05,** p<0.01, and ***p<0.001.

Figure 3. MiR-217 targets WASF3 in osteosarcoma cells.

(A) The sequences of miR-217 binding sites within the human WASF3 3′UTR and schematic reporter constructs, in this panel, WASF3-WT represent the reporter constructs containing the entire 3′UTR sequences of WASF3. WASF3-MUT represent the reporter constructs containing mutated nucleotides. (B) The analysis of the relative luciferase activities of WASF3-WT, WASF3-MUT in 293T cells. The error bars are derived from triplicate expriments. (C) qRT-PCR analysis of WASF3 mRNA expression in MG-63 cells after treatment with miRNA mimics or scramble or no transfection. The expression of WASF3 was normalized to GAPDH. (D) Western blot analysis of WASF3 expression in MG-63 cells transfected with miR-217 mimics or scramble or no transfection. GAPDH was also detected as a loading control. All data uses t test and is shown as mean ±SEM.

Figure 4. Restore WASF3 expression impairs miR-217-induced inhibition of proliferation and invasion in osteosarcoma cells.

(A) Western blot analysis of WASF3 in MG-63 cells co-transfectedwith either miR-217 mimic or scramble and 2.0 µg pCDNA-WASF3 orpCDNA empty vector. (B) The relative WASF3 protein was showed with different combinations. The signal in each lane was quantified using ImageJ software and the ratio of WASF3 to GAPDH was determined. (C) Cell growth curves in MG-63 cells transfected with different combinations using CCK-8 analysis. (D) The relative ratio of wound closure per field with different combinations is shown. (E) Transwell analysis of MG-63 cells treated with different combinations. The invasive cells per field is shown right. (F) Wound healing assays of MG-63 cells after treatment with different combinations. (G) Western blot analysis of WASF3 expression in 8 miR-217 down-regulated osteosarcoma tissues and their pair-matched nontumor tissues. GAPDH was also detected as a loading control. All data uses t test and is shown as mean ±SEM. *p<0.05,** p<0.01,***p<0.001.