microRNA-124 Inhibits Migration and Invasion by Down-Regulating ROCK1 in Glioma

<div><p>Background</p><p>The extraordinary invasive capability is a major cause of treatment failure and tumor recurrence in glioma, however, the molecular and cellular mechanisms governing glioma invasion remain poorly understood. Evidence in other cell systems has implicated the regulatory role of microRNA in cell motility and invasion, which promotes us to investigate the biological functions of miR-124 in glioma in this regard.</p><p>Results</p><p>We have found that miR-124 is dramatically downregulated in clinical specimen of glioma and is negatively correlated with the tumor pathological grading in the current study. The cells transfected by miR-124 expression vector have demonstrated retarded cell mobility. Using a bioinformatics analysis approach, rho-associated coiled-coil containing protein kinase 1 (ROCK1), a well-known cell mobility-related gene, has been identified as the target of miR-124. A dual-luciferase reporter assay was used to confirm that miR-124 targeted directly the 3′UTR of ROCK1 gene and repressed the ROCK1 expression in U87MG human glioma cell line. Furthermore, experiments have shown that the decreased cell mobility was due to the actin cytoskeleton rearrangements and the reduced cell surface ruffle in U87MG glioma cells. These results are similar to the cellular responses of U87MG glioma cells to the treatment of Y-27632, an inhibitor of ROCK protein. Moreover, a constitutively active ROCK1 in miR-124 over-expressed glioma cells reversed the effects of miR-124. Our results revealed a novel mechanism that miR-124 inhibits glioma cells migration and invasion via ROCK1 downregulation.</p><p>Conclusions</p><p>These results suggest that miR-124 may function as anti-migration and anti-invasion influence in glioma and provides a potential approach for developing miR-124-based therapeutic strategies for malignant glioma therapy.</p></div

Evaluation of biological functions of miR-124 in transfected HEK293ET and U87MG cells.

<p>(A) The expression level of miR-124 was measured using qRT-PCR after miR-124 transfected HEK293ET and U87MG cells for 48 h. Data presented are mean values of three independent experimental results and compared with the level of miR-124 obtained in mock control (Lipofectamine2000 blank) that is normalized to 1. (B) Wound-healing assay of U87MG glioma cells transfected with either control or the miR-124 expression vector, respectively.</p

qPCR assays of miR-124 expression levels in glioma tissue samples.

<p>The expression level of miR-124 was downregulated significantly in high grade human glioma tissues (five grade III and three grade IV) than that in low grade human glioma tissues (three grade I and five grade II) determined using qRT-PCR.</p

ROCK1 is target of miR-124.

<p>(A) Illustration of the predicted miR-124-binding sequences in the 3′UTR region of ROCK1. (B) The calculated free energy for hybridization of the ROCK1 3′UTR and miR-124 (Red color: ROCK1, Green color: miR-124). (C) Homology analysis of the 3′UTR sequences of 13 different species recognized by miR-124 seed sequence. (D) Luciferase analysis in HEK293ET cells. The assay was repeated three times with each assay being performed in three wells, and similar results were obtained each time. (E) qRT-PCR assay of ROCK1 levels treated with either pcDNA3.1 or pcDNA3.1-miR-124 for 48 h, compared with mock control in U87MG cells. (F) Western blot analysis of ROCK1 expression treated with either pcDNA3.1 or pcDNA3.1-miR-124 for 72 h, compared with mock control in U87MG cells.</p

Reintroduction of ROCK1 rescues the miR-124-induced invasion inhibition.

<p>(A) Stress fiber staining in U87MG glioma cells treated with miR-124 expression vector along with p160^ROCKΔ3 or control pCAG-myc (Scale bars: 20 µm). (B) Scanning electron microscopy of U87MG cells. (C) In vitro invasion assay. Invasive cells were stained and the average number of cells was counted at random six fields of vision. The data was an average value of three independent experiments. (Magnification: 100×; scale bars: 100 µm).</p

Identification of biological function of miR-124 in U87MG cells.

<p>(A) In vitro invasion assay. Invasive cells were stained and the average number of cells was counted at random six fields of vision. The data was an average value of three independent experiments. (Magnification: 100×; scale bars: 100 µm) (B) Stress fiber staining in U87MG glioma cells (Scale bars: 20 µm). (C) Scanning electron microscopy of U87MG cells treated with miR-124 expression vector or control and Y-27632 (Scale bars: 10 µm).</p

Proposed model of miR-124 function in glioma development and progression.

<p>The target of miR-124 in bold fonts was confirmed in this study.</p

Effects of other analog substitutions on <i>Eco</i>RI cleavage.

<p>(A) The effects of 2′-FNA substitution at different positions on the reaction initial velocity. (B) Comparison of initial reaction velocities of different nucleotide derivatives at G1 position. (C) Comparison of initial reaction velocities of different nucleotide derivatives at A2 position.</p

Effects of 2′-OMeN substitution on <i>Eco</i>RI cleavage.

<p>(A) Fluorescence intensity changes of the 2′-OMeN-modified <i>Eco</i>RI sequences with respect to reaction time at the <i>Eco</i>RI concentration of 1.2 unit/µl. (B) Initial reaction velocities calculated based on the fluorescence curves in Fig. 5A.</p

Illustration of three neokinks in <i>Eco</i>RI-bonded duplex.

<p>Illustration of three neokinks in <i>Eco</i>RI-bonded duplex.</p