MiR-145 reduces ADAM17 expression and inhibits in vitro migration and invasion of glioma cells

Abstract

MicroRNAs are important regulators of gene expression and have been suggested to play a key role in tumorigenesis. In this study, we show that miR-145 is significantly downregulated in glioma cell lines compared to normal brain tissue and negatively regulates tumorigenesis. Restoration of miR-145 in glioma cells significantly reduced in vitro proliferation, migration and invasion. Also, overexpression of miR-145 reduced ADAM17 and EGFR expression. In addition, we tested the hypothesis that the miR-145-mediated suppression of cell proliferation, migration and invasion is, at least in part, due to silencing of ADAM17 and EGFR gene expression. Using luciferase reporters carrying the 3'-untranslated region of ADAM17 combined with western blotting, we identified ADAM17 as a direct target of miR-145. Collectively, these results suggest that as a tumor suppressor, miR-145 inhibits not only tumor proliferation, but also cell migration and invasion, and warrants further investigation.

Figure 1

mir145 expression levels in glioma cell lines. (A) Total RNA was isolated from the normal brain tissue and glioma cells of U87, U251n, HF66, and T98G and real-time PCR was employed to analyze the expression of miR-145. The relative expression of miR-145 was expressed as the ratio of the expression level of normal brain tissue. ^*P<0.01, as compared with normal brain tissues. (B) Glioma cells were transfected with miR-145 mimic for 72 h, and then collected for real-time RT-PCR. The expression of miR-145 was significantly increased in U87 and U251n, as compared with non-transfected cells. ^*P<0.01, as compared to glioma cells without transfection of miR-145.

Figure 2

Overexpression of miR-145 reduces glioam cell proliferation in vitro. Glioma cells were transfected with miR-145 and negative control. After incubation for 72 h, cell proliferation rates were analyzed by BrdU assay. ^*P<0.01, as compared to control and negative control.

Figure 3

Effect of miR-145 overexpression on cell migration and invasion in U251n and U87. (A) Wound healing assay was performed to evaluate the effects of miR-145 on glioma cell migration. U251n cells were transfected with miR-145 at a final concentration of 300 nM. When the cell confluence reached ~90% at 72 h post-transfection, an artificial homogeneous wound was made as described in Materials and methods. The experiments were preformed in triplicate and the representative images of photographs at 0 and 24 h post-wounding are shown at magnification ×100. ^*P<0.01, as compared to control and negative control. (B) Effect of miR-145 overexpression on glioma cell invasion. Glioma cells transfected with miR-145 or negative control were plated on Matrigel-coated membranes in the upper chamber of transwells. After incubation for 24 h, non-invading cells on the upper surface of the membrane were removed and the invasive cells on the lower surface were stained with cell tracker. The stained invasive cells were photographed under a fluorescence microscope (magnification ×200). ^*P<0.01, as compared to control and negative control. (C) To confirm the effect of miR-145 overexpression on glioma cells invasion, we employed the cell line U87. U87 cells were also transfected with miR-145 or negative control, and then analyzed with Matrigel-coated membranes chamber invasion assay. ^*P<0.01, as compared to control and negative control.

Figure 4

ADAM17 is a predicted target of miR-145 for post-transcriptional repression. (A) The miR-145 seed sequences and their predicted binding sites in the ADAM17 3′-UTR are shown. The highlighted region representing the binding site was predicted by Targetscan. (B) The mRNA expression of ADAM17 and EGFR in glioma cells. Overexpression of miR-145 did not decrease the mRNA expression of EGFR and ADAM17. (C) MiR-145 targets the predicted binding site within the ADAM17 mRNA 3′-UTR, decreasing luciferase in U87 and U251n cells transfected with a luciferase reporter vector containing the predicted binding site. Mutant-ADAM17, vector containing mutated binding site; wild-type-ADAM17, vector containing predicted binding site. ^*P<0.01 compared to U87 or U251n transfected with vector containing a mutated ADAM17 site and miR-145. (D) The protein expression of ADAM17 and EGFR in U251n cells. Western blot analysis was employed to test the expression of ADAM17 and EGFR in glioma cells. β-actin was used as an internal standard.