Study On Expression Of Arp2/3in Glioma And Its Role In The Motility Of Glioma Cells

Glioma is the the most common brain tumor and glioblastoma is the most common primary malignant brain tumor, accounting for approximately12-15%of all intracranial neoplasms and60-75%of astrocytic tumors. Despite the recent advances in therapies for glioblastoma, including surgical resection followed by radiotherapy with concomitant and adjuvant chemotherapy, the2-year survival rate of most patients with glioblastoma is only approximately25%. Though maximal safe surgical resection is given,80%of patients experience tumor recurrence within2cm of the resection margin. The biological features of aggressive infiltration into adjacent tissues and replasing easily usually make glioblastoma incurable. Therefore, it is very necessary to discover novel therapeutic strategies, aimed at effective control of the tumor recurrence and progress. One of such approaches is to control progression, invasion, and metastasis of malignant glioma cells.Arp2/3complex as an important downstream effector of small Rho GTPases-WAVE could nucleate actin polymerization and play an indispensable role in the cell motility. When cell encounter extracellular stimuli (such as chemokines), Arp2/3complex will receive the signal transduction from Rho GTPases-WAVE and it will bind the existing actin mother filaments and initiate the formation of daughter filaments. With the formation of dendritic actin filaments, they push against the membrane to form the protrusion, which provide force for the cell motility.This study was mainly designed to detect the expression of Arp2/3complex in glioma, and to find the role of Arp2/3complex in the formation of lamellipodia as well as in the motility of glioma cells, including two parts:1. To observe the expression of Arp2/3in human glioma and analyze the correlation of Arp2/3expression with the pathological grade of glioma,50specimens of glioma with different pathological grades and8control brain tissues acquired from surgeries for temporal lobe epilepsy were collected to perform immunofluorescence histochemistry and western blot analysis. In the immunofluorescence analysis for the Arp2/3expression in different specimens, the data revealed that the expression of Arp2/3complex was negative or just weakly positive in control brain tissues, but its fluorescence intensity increased with elevated pathological grades in the tumor tissues(F=83.538, P=0.000).Semi-quantitative assessment of Arp2/3complex levels by western blot indicated that the level of Arp2/3was15.69±1.04%in control brain tissues (n=8),31.17±4.30%in WHO Grade I gliomas (n=8),40.51±2.12%in WHO Grade Ⅱ gliomas (n=14),48.68±2.69%in WHO Grade Ⅲ gliomas (n=10), and55.42±3.45%in WHO Grade Ⅳ gliomas (n=18). The expression of Arp2/3in the glioma specimens was significantly higher than in control brain tissues (P<0.05) and a positive correlation was found between the expression of Arp2/3and the malignancy of glioma specimens (r=0.686, P=0.02).2. The role of Arp2/3complex in the lamellipodia formation and motility of glioma cells.①To investigate the role of Arp2/3in the lamellipodia formation and motility of glioma cells, we inhibited the Arp2/3activity by specific Arp2/3inhibitor, CK666. The immunofluorescence assay and wound-healing assay were conducted to identify the relationship between lamellipodia and the movement direction. The MTT assay was used to measure the effect of specific Arp2/3inhibitor on cell survival. We also performed immunofluorescence assay to evaluate the location of Arp2/3, and the inverted phase contrast microscope for the morphological changes of glioma cells, while the wound-healing assay and transwell invasion assay were employed to evaluate migration and invasiveness capability of glioma cells in vitro. The data demonstrated that lamellipodia of glioma cells faced to the movement direction, and Arp2/3localized in the actin-rich lamellipodia. Inhibition of Arp2/3led to the disappearance of lamellipodia, and significant reduction of migration and invasion capability of glioma cells (P<0.05).②As a matter of fact, there is no any chemical inhibitor that is completely to the target it claims. To validate the role of Arp2/3in the lamellipodia formation and motility of glioma cells, we further used a synthetic siRNA to knock down the Arp2/3in U251glioma cells. Reverse transriptase-polymerase chain reaction (RT-PCR) and western blot were performed to test the efficiency of RNAi. We also performed immunofluorescence assay, the wound-healing assay and transwell invasion assay to evaluate the effect of Arp2/3 knockdown on lamellipodia formation and motility of glioma cells. The data demonstrated that no obvious lamellipodia formation was found and the capability of cell motility declined sharply in Arp2/3knocked down U251glioma cell by siRNA (P<0.05).The conclusions at this study were drawn as follows:1. The Arp2/3complex expressed negatively or just weakly positively in control brain tissues, but its expression was positive in glioma and the expression rate was positively correlated with the tumor pathological grades.2. The lamellipodia of glioma cells faced to the movement direction and Arp2/3was co-localized with actin in cytoplasm. Inhibition of Arp2/3by the specific chemical inhibitor or knocking down of Arp2/3by RNAi could inhibit the formation of lamellipodia and cell motility obviously.