Study On Expression Of Rac1 In Gliomas And Its Underlying Mechanism In Promoting The Motility Of Glioma Cells

Glioblastoma multiforme(GBM), the most common and malignant central nervous system(CNS) tumors in humans, is one of human treatment-resistant and poorest prognosis tumors. Despite the use of standardized treatment measures, there has been minor improvement for glioma patient therapy. Although maximal safe surgical resection is given, patients of glioblastoma undergo tumor recurrence within2 cm of the tumor margin. Histological evidence has shown that glioma cells always disperse along the orientation of thin, elongated anatomic structures, such as white matter fibers, capillaries, and unmyelinated axons. The characteristics of invasion and migration are the major cause for the poor prognosis of glioblastoma. Therefore, more and more people realize that the targeted inhibitory of the molecular regulators of glioma invasion and migration will provide a new way for the treatment of glioma.The invasion and migration of glioma cells is an integrated and complex multistep process. The invasion of glioma cells requires four basic steps: ○1 The invading tumor cells detached from the tumor entity; ○2 The tumor cell adhesion to extracellular matrix(ECM);○3 Proteolytic breakdown of ECM;○4 Cell movement and contraction. Cell migration plays a crucial role among them. Cell migration is controlled by a variety of signaling molecules, among them, the actin cytoskeleton and its regulatory proteins are most important. The full name of Rac1 is Ras-related C3 botulinum toxin substrate1, and Rac1 gene is 29 kb, containing 7 exons, located on human chromosome 7p22. Recent studies have found that Rac1 was essential for Ras-induced malignant transformation. Rac1, a member of Rho family, is found in two forms, a GDP-bound inactive form and a GTP-bound active form. Rac1 functions as molecular switches in a wide range of signal transduction pathways of eukaryotic cells, which translates lots of extracellular signals into intracellular events that ultimately induce changes in the organization of the actin cytoskeleton. As a molecular switch, Rac1 is involved in regulating a number of cellular functions,including cell proliferation, apoptosis, migration and invasion of tumor cells. TheV formation of cell lamellipodia and focal adhesion turnover are two key steps of cell migration, which are crucial for cell migration. Rac1, a regulatory factor in the process of tumor cells invasion and migration, is not only involved in regulating the formation of cell lamellipodia, but also implicated in regulating the turnover of focal adhesions. Previous studies showed that overexpression of Rac1 correlates with progression of epithelial ovarian cancer, gastric carcinoma, and hepatocellular carcinoma.This study was aimed to detect the expression of Rac1 in glioma and to investigate the underlying mechanisms of Rac1 increasing the motility of glioma cells,including three parts:1. To detect the Rac1 expression of human gliomas and analyze the relationship between Rac1 expression and the pathological grade of glioma. We selected 8 control brain tissues and 40 specimens of glioma with different pathological grades as the experimental object. To determine Rac1 expression in human gliomas, both immunohistochemistry and western blotting were employed. The data of immunohistochemistry demonstrated that the normal brain tissues did not express Rac1, but the level of Rac1 protein in gliomas was elevated and positively correlated with the malignancy of gliomas(F=105.225, P=0.000).The results of western blotting showed that the levels of Rac1 were 0.093±0.025 in control brain tissues(n=8). The levels of Rac1 protein in WHO grade II(n=8), III(n=12) and IV(n=20) were 0.22±0.031, 0.38±0.031 and 0.61±0.025, respectively.Compared with nonneoplastic brain tissues, the levels of Rac1 protein in glioma were markedly elevated and positively correlated with the tumor grade of glioma(r=0.92,P=0.012).2. The role of Rac1 in the migration and invasion of human glioma cells. To investigate the role of Rac1 in the migration and invasion of human glioma cells, we inhibited Rac1 activity with the Rac1-specific small molecule inhibitor NSC23766.U251 and SNB19 glioma cells were employed and assigned into two groups: control group and NSC23766 treated group. The GST pull-down assay was used to detect the Rac1 activity.The MTT assay was employed to measure the effect of NSC23766 on cell survival. We used the transwell invasion assay and wound-healing assay toevaluate the motility ability of glioma cells in vitro. The results indicated that NSC23766 significantly inhibited the Rac1 activity( P<0.01) and remarkedly impaired the migration and invasion of glioma cells(P<0.01).3. The underlying mechanisms of Rac1 enhancing the motility of glioma cells. In this part, both Rac1-specific inhibitor NSC23766 and Rac1-specific si RNA were employed to investigate the the underlying mechanism of Rac1 promoting the motility of glioma cells. Immunofluorescence was used to localise the expression of Rac1 in glioma cells and observe the change of cell morphology and the distribution of Rac1 and LIMK1 in glioma cells. The expression of Rac1 in control, si RNA-NC and Rac1 si RNA groups was detected by western blotting. The expression of P-LIMK1(Thr508), LIMK1, MMP-2 and MMP-9 in different group was determined by western blot analysis. The migratory and invasive abilities of human glioma cells in control group, Rac1 si RNA group and LIMK1 si RNA group were evaluated by wound-healing assay and transwell invasion assay, respectively. The lamellipodia became smaller or never spreaded in NSC23766 treated group,whereas it was kept“normal”in control group. NSC23766 also inhibited focal adhesion turnover, which is required for cell migration. In addition, NSC23766 markedly impaired the the phosphorylation of LIMK1(P<0.01). The levels of Rac1 protein in U251/SNB19 cells were remarkably down-regulated by Rac1 si RNA(P<0.01). Accordingly, silencing of Rac1 expression acquired the similar results. We observed that Rac1 and LIMK1 were co-localized in the lamellipodia of glioma cells by immunofluorescence. In comparison with the control group, the migration and invasion capacities of glioma cells was markedly inhibited in Rac1 si RNA group and LIMK1 si RNA group(P<0.01).Additionally, there was down-regulation of matrix metalloprotease-2 and-9expression in NSC23766 treated cells, which coincided with decreased invasion in vitro(P<0.01).Conclusions:1.The Rac1 protein levels of glioma tissues was remarkedly increased compared with those in control brain tissues and positively correlated with the pathological grading of glioma.2. Rac1 plays an important role in the migration and invasion of glioma cells.3.Rac1 is implicated in the control of glioma cell migration and invasion by regulating lamellipodia formation, the turnover of focal adhesions and matrix metalloprotease-2 and-9 expression.