Study On Expression Of Cortactin In Glioma And Its Role In The Motility Of Glioma Cells

Glioma is the the most common brain tumor, accounts for about2%of the adult body tumors. Statistics show that more than half of them are the most highly malignant glioma-glioblastoma (GBM), it is known as aggressive growth, microsatellite, the tumor cells infiltrating in the normal brain parenchyma, is one of human treatment-resistant tumor. Despite the recent advances in therapies for glioblastoma, including surgical resection followed by radiotherapy with concomitant and adjuvant chemotherapy, the5-year survival rate of patients with glioblastoma is only approximately10%and the survival rate is lower for elderly patients.Cortactin, a multi-domain protein, can regulate actin polymerization and depolymerization through a variety of approaches and can affect the formation and sustain of cell "movement organs"-lamellipodia. It possess a multi-domain structure consisting of an acidic domain at the amino terminus (NTA), followed by six complete and one partial tandem repeating segments, an a helical region which is proline-rich, and a Src homology SH3domain located at the carboxyl terminus.The NTA domain of cortactin can interact with the Arp2/3complex to regulate actin polymerization and depolymerization. The SH3domain of cortactin can interact with N-WASP to enhance the above process. The over expression of cortactin exist in many cancers, including head and neck squamous cell carcinoma (HNSCC), oral squamous cell carcinoma and prostate cancer, but its relationship with gliomas has not be confirmed.This study was designed to investigate the role of cortactin in the infiltrative behavior of glioma cells and the potential mechanism of cortactin promoting the migration and invasion of glioma cells, including three parts:1. To evaluate the expression of cortactin in human gliomas and non-tumor brain tissues,40specimens of glioma with different pathological grades and8non-tumor brain tissues were collected for study. Immunohistochemistry and western blotting analysis were implemented for them. In the immunohistochemistry analysis for cortactin expression, the data revealed that cortactin expressed weakly in non-tumor brain tissues, but strongly expressed in gliomas and the expression level of coractin was positively correlated with the malignancy of gliomas.Relative to β-actin, the level of cortactin in tissue specimens was18.20±2.52%in non-tumor brain tissue (NB, n=8),36.20±3.34%in WHO grade II (n=15),51.40±4.30%in WHO grade Ⅲ (n=9) and59.60±4.31%in WHO grade IV (n=16). The expression of cortactin in the glioma specimens was significantly higher than in non-tumor brain tissue (P<0.01) and positively correlated with the malignancy of glioma specimens (r=0.912, P=0.00).2. The role of cortactin in the motility of glioma cells. U251, LN229and SNB19glioma cells were employed and assigned into three treated groups:siRNA-cortactin group (transfected by siRNA specific to cortactin), siRNA-NC group (transfected by negative control RNA sequence) and siRNA-N group (transfected by empty vector) in vitro, respectively. The expression of cortactin in different treated group was detected by QRT-PCR and western blotting. The migration and invasion of glioma cells under different treatments were evaluated by wound-healing assay and transwell-chamber invasion assay respectively. Result:The cortactin expression in glioma cells was inhibited dramatically (P<0.05), and their migration and invasion ability was also impaired significantly after treated with siRNA (P<0.05) compared with the other two groups.3. The potential mechanism of cortactin promoting the migration and invasion of glioma cells. The lamellipodia of glioma cells after treatments were observed by immunofluorescence (IF), and lamellipodia changing with time were photoed by inverted microscope. The distribution of cortactin and Arp2/3complex in glioma cells were detected by IF. The combination of cortactin and Arp2/3in glioma cells were detected by Co-immunoprecipitation(Co-IP). Result:The size and persistence time of lamellipodia was reduced after inhibiting cortactin expression in glioma cells. Cortactin and Arp2/3complex were co-localized in the front of glioma cells, where actin was polymerized and lamellipodia formed. The combination between cortactin and Arp2/3complex in glioma cells was demonstrated by Co-IP.The conclusions at this study were drawn as follows:1. The expression of cortactin in the glioma specimens was significantly higher than in non-tumor brain tissue and positively correlated with the malignancy of glioma specimens.2. The ability of migration and invasion was declined siginificantly after the glioma cells were treated with siRNA specific to cortactin.3. The size and persistence time of lamellipodia was reduced after inhibiting cortactin expression in glioma cells. Cortactin and Arp2/3complex were co-localized in the front of glioma cells, where actin was polymerized and lamellipodia formed. The combination of cortactin and Arp2/3complex in glioma cells were demonstrated by Co-IP.