Inhibition of tumor cell migration and invasion through knockdown of Rac1 expression in medulloblastoma cells

Abstract

Medulloblastoma is one of the common malignant brain tumors in children or young adults and its overall disease-free 5-year survival rate is approximately 50% due to tumor progression, invasion, and metastasis. This study aimed to determine whether one of Rho GTPases, Rac1 can affect the morphology, motility, and invasion of medulloblastoma cells through knockdown of Rac1 expression. Medulloblastoma Daoy cells were used to manipulate Rac1 expression using Rac1 shRNA, Rac1N17, and Rac1L61 constructs. Reverse transcriptase-PCR and western blot were used to detect expression of Rac1 mRNA and protein, respectively. Invasion and migration assays were performed to assess invasion and migration capacity of Daoy cells, respectively. The data showed that Rac1 mRNA and protein were overexpressed in Daoy cells. Deletion of Rac1 decreased the cross-linked actin network and pseudopodia and also inhibited the number of migration cells migrated or invaded to the other side of the filter compared to control cells. These data indicated that the invasion and migration in Daoy cells were inhibited by deletion of Rac1, and suggest that targeting Rac1 by Rac1 shRNA may further be evaluated and used as a potential anticancer strategy to treat medulloblastoma.

Fig. 1

Expressions of Rac1 mRNA and protein in Daoy cells. Rac1 mRNA (a) and protein (b) were overexpressed in both cell lines (Daoy and U251). Deletion of Rac1 inhibits the expression of Rac1 protein in Daoy cells (c)

Fig. 2

The roles of Rac1 in Daoy cells cytoskeleton and lamellipodia formation. Confocal micrograph shows deletion of Rac1 reduces serum-induced cross-linked actin network staining with FITC-conjugated phalloidin in Daoy cells (b), while cross-linked actin network can be seen in Daoy cells of control (a). Deletion of Rac1 inhibits serum-induced lamellipodia and pseudopodia formation (d, f), but this phenomenon (middle arrows) can be seen in Daoy cells treated with empty plasmid vector (c, e), All images were at 400× magnification

Fig. 3

Tumor cell migration assay. The Daoy cells were grown and subjected to transient gene transfection with Rac1-shRNA (a and b), Rac1N17 (c and d), Rac1L61 (e and f) or vector-only control (g and h), respectively. After the cells reached almost confluence and wound hearing assay was performed and photos were taken at 0 and 24 h. All images were at 200× magnification. The level of tumor cell migration into the wound scratch area was quantified as percentage of control. * P < 0.05; ** P < 0.01

Fig. 4

The level of cell migration into the wound scratch was quantified as the percentage of wound healing and compared against that of control Daoy cells at each time point, * P < 0.05; ** P < 0.01 (a). Deletion of Rac1 inhibited Daoy cells invasion. Daoy cells were transfected with Rac1-shRNA, Rac1N17, Rac1L61, and Control, respectively, and cells migrating into the underside of the chamber were stained with crystal violet and counted under 100× Magnification. Bars represent ±SE. Data are representative of three independent experiments, * P < 0.05; ** P < 0.01 (b)