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. 2016;23(5):257-66.
doi: 10.3727/096504016X14562725373752.

MicroRNA-20b Downregulates HIF-1α and Inhibits the Proliferation and Invasion of Osteosarcoma Cells

Ming Liu  1 , Dan Wang, Ning Li
Affiliations

MicroRNA-20b Downregulates HIF-1α and Inhibits the Proliferation and Invasion of Osteosarcoma Cells

Ming Liu et al. Oncol Res. 2016.

Abstract

Osteosarcoma (OS) is the most common malignant primary bone tumor disease. HIF-1α was predicted to be the target gene of microRNA-20b (miR-20b). The present study was designed to illustrate the effect of miR-20b in regulating osteosarcoma via targeting HIF-1α. In this study, we found that the expression of HIF-1α was significantly increased, while miR-20b obviously decreased in OS patients and OS cell lines compared with healthy controls. Moreover, the luciferase report confirmed the targeting reaction between miR-20b and HIF-1α. Additionally, the overexpression of miR-20b suppressed the invasion and growth of both MG63 and U2OS cells, and inhibited the expressions of HIF-1α and VEGF pathway proteins, while the inhibition of miR-20b led to the reverse results. Furthermore, the overexpression of HIF-1α affected the suppression effect of miR-20b in MG63 cells, indicating that miR-20b suppresses the tumor cell process via inhibiting the expression of HIF-1α. Taken together, our results suggest that the upregulation of miR-20b affects the expression of HIF-1α, downregulates the VEGF pathway proteins, and suppresses cell invasion and proliferation rate. These results provide a potential therapeutic strategy for osteosarcoma.

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Figures

Figure 1
Figure 1
Inverse level of miR-20b and HIF-1α in OS patients and healthy controls. (A) The expression levels of miR-20b in OS patients and healthy controls were measured by quantitative real-time PCR (qRT-PCR). (B) The mRNA expression of HIF-1α in OS patients and healthy controls was assessed by qRT-PCR assays. (C) The correlation between miR-20b expression levels and HIF-1α mRNA expression levels in OS patients. (D) The expression levels of miR-20b in OB cell lines and OS cell lines were measured by quantitative real-time PCR (qRT-PCR). (E) The mRNA expression of HIF-1α in OB cell line and OS cell lines was assessed by qRT-PCR assay. (F) Western blotting assay was used to detect the expression profile of HIF-1α in OB and OS cell lines. Relative protein expression was quantified using Image-Pro Plus 6.0 software and normalized to β-actin. Data are represented as the mean ± SD of three experiments. *p < 0.05 versus healthy controls group, #p < 0.05 versus OB group.
Figure 2
Figure 2
Effect of miR-20b on the invasion and proliferation of MG63 and U2OS cells. MG63 and U2OS cells were transfected with miR-20b mimic, mimic control, miR-20b inhibitor, or inhibitor control, respectively. (A) The relative miR-20b levels in MG63 and U2OS cells were measured by qRT-PCR. (B) The number of invading MG63 and U2OS cells. The proliferation of MG63 (C) and U2OS (D) cells was determined at the indicated time points by MTT assays. All experiments were repeated three times with three replicates. *p < 0.05 compared to mimic control group; #p < 0.05 compared to inhibitor control group.
Figure 3
Figure 3
The target relationship between miR-20b and HIF-1α. The target gene was predicted by TargetScan database and identified by luciferase activity report. (A) The wild-type and the mutant HIF-1α 3′-UTR contained the target sequence of miR-20b. (B) MG63 cells were cotransfected with miR-20b mimic or mimic control luciferase reporter vectors containing wild-type (wt) or mutant (mut) HIF-1α 3′-UTR. (C) A similar luciferase assay was performed in MG63 cell lines treated with miR-20b inhibitor or inhibitor control. Luciferase activity was represented as firefly luciferase normalized to renilla luciferase. Data are represented as the mean ± SD of three experiments. *p < 0.05 versus other three groups.
Figure 4
Figure 4
miR-20b regulates VEGF pathway via targeting HIF-1α. The MG63 and U2OS cells were transfected with the miR-20b mimic, mimic control, miR-20b inhibitor, or inhibitor control, separately. (A) The protein expression levels of HIF-1α and VEGF pathway genes in MG63 cells were measured by Western blotting. (B) A similar Western blotting assay was performed in U2OS cells. (C) Relative protein expression levels in MG63 cells were quantified using Image-Pro Plus 6.0 software and normalized to β-actin. (D) Relative protein expression in U2OS cells. Data are represented as the mean ± SD of three experiments. *p < 0.05 versus mimic control, #p < 0.05 versus inhibitor control.
Figure 5
Figure 5
Overexpression of HIF-1α offsets the suppression effect of miR-20b in MG63 cells. Cells were treated with miR-20b mimic or mimic control with or without HIF-1α overexpression vector. (A) HIF-1α, VEGF, Cdc42, P38, and HSP27 expressions were detected by Western blotting. (B) Relative protein expressions in MG63 cells were quantified using Image-Pro Plus 6.0 software and normalized to β-actin. (C) The number of invading MG63 cells. (D) MTT assay was employed to examine proliferation rates of MG63 cells under similar treatments. The data are shown as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01 indicates significantly different.
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