Primary Study Concerned The Biologcal Influence On Medulloblastoma Performed By RNA Interference Of Racl Gene

BACKGROUND & OBJECTIVEMedulloblastoma (MB) is one of the most common malignant brain tumor in children, which belongs to a neuroepithelial tumor with invasive growing characteristics and account for about 1/4 children's malignant brain tumors. The peak incidence of medulloblastoma is at 5 to 10 years old, and the morbidity of medulloblastoma is inferior to that of astrocytoma in children. In adults,80% medulloblastoma happens at 21-40 years old. The incidence of men is more prevalent than that of women. Medulloblastoma belongs to one of neuroectodermal tumors, is generally believed that it possesses the double differentiation characteristics of the neurons and glial cells. The WHO classification of medulloblastoma in 2000 is divided into classic-type, connective tissue-type, large cell-type, myeloid myoblasts-type and melanoma-type. Medulloblastoma is highly invasive and metastatic, so it has high mortality, poor prognosis and the low cure rate. In recent years, because of the advances in neurological surgery techniques and radiotherapy techniques, the application of chemotherapy drugs, the continued standardization of treatment, the mortality and disability of medulloblastoma have been obviously reduced. At present,5-year survival rate of medulloblastoma is at 50%～70%,10-year survival rate of that is at 30%～50%. Even though great progress has been made, there are still a considerable great number of patients died of tumor's recurrence or metastasis.The course of tumor's invasion or metastasis is such a complicated multi-step process, which is required to breakthrough a number of barriers composed of the extracellular matrix and basement membrane structure; Cancer cells detach from the primary tumor, then pass through the epithelial basement membrane and invade into the surrounding matrix and the neighboring organizations, ultimately enter the blood or lymphatic vessels. Some of them evade the immune surveillance so that they could set up a new exuberant proliferation cell-colony in remote parts by exuding from the vascular. Each process of those is very complex, the whole process is subject to a precise control of cross-linked signal transduction network which were composed of a number of interrelated signal transduction pathways. It is perceived from the process of tumor invasion and metastasis, that the alternate changes of adhesion and migration of tumor's activity are essential to the tumor invasion and metastasis.Rho proteins (including Rho, Racl, Cdc42) which have the GTP-enzyme activity, play an important role in the development of tumors. Rho proteins and its downstream effective molecules are involved in the malignant tumor transformation, invasion and metastasis and angiogenesis, through a variety of signal transduction pathway. So Rho proteins have become a very promising new target for tumor treatment. Racl (Ras-related C3 botulinum substrate protein 1) is one of important members of Rho protein, DNA of which is located on the 7th of human short arm of chromosome 2 with 2 zones (7p22), molecular weight of which is 25KD, and Genebank NO. of which is NM₀06908. The protein which the Rac1 gene encoded belonged to the Ras superfamily of small GTP-binding protein, the role of which is to regulate the cells'various functions, including control of cell growth, cytoskeletal reorganization and activation of protein kinase. Later, some studies have decleared that Racl is also an important role in cell migration, cell-cell or cell-extracellular matrix adhesion, cell division and cell anoikis. There are three types of Rac protein, Rac1,2 and 3 in human genome. These three proteins were highly homologous, However, those are different in transcription regulation and organization of distribution. Besides Racl is distributed widely, Rac2 existed in hematopoietic tissues, Rac3 is at high expression in brain tissue, but is at low expression in other tissues. There are two forms in Racl protein, Racl-GDP (inactivation status), Racl-GTP (activated) respectively. As a signal converter or molecular switches in cell signal transduction pathway, Racl protein has an biological effect on cytoskeleton or its target protein. Racl is the upstream regulatory molecules of Tiaml, that is, T lymphoma invasion and metastasis-inducing factor, and its downstream effectors is PAK1, or P21-activated kinase 1. Of course, the different roles of Racl are regulated by different signal transduction pathways. Salhia et al. used RNAi methods to interfere the expression of Racl, then the changes of cell morphology, migratory capacity and invasiveness of astrocytoma cell were found after the silence of Racl gene; Amanda et al. found that the invasiveness of both glioma and breast cancer were significantly weakened by silence of Racl gene; Klooster et al. investigated the molecular mechanism of PAK1, which was regulated by Racl in further studies. They found that the activation of Racl manipulated actin polymerization, prominent membrane and thus led the movement of HEK293 cells.RNA interfered technique (RNAi) was the means to insert a section of double-stranded RNA (dsRNA) into cells. Host cells responded quickly to this dsRNA, then the endonuclease Dicer in the cytoplasm cut dsRNA into a specific length and structure of the small fragment RNA, which also was called siRNA. These siRNA blocked specific gene expression efficiently in vivo through degradation of the relevant specific mRNA genes, so that the cells could appear specific gene deletion phenotype to achieve some experimental or therapeutic purposes. As far, few literatures concerned on the Racl and medulloblastoma had been reported. In order to elucidate the ralationship between Racl protein and the tumor invasion, metastasis in medulloblastoma, medulloblastoma tissue and its cell line were taken for research and gliomas were saved as the control group. First of all, we explored Racl protein expression in different levels of glioma, medulloblastoma and normal brain tissues in order to initially define the possible relations between Racl protein and the tumor occurrence and development. Small interfering RNA (siRNA) technique was performed to construct recombinant vector to silence Racl gene in Daoy cell (medulloblastoma cell line). So that we could investigate effects of Racl's down-regulation on various aspect of medulloblastoma, such as tumor migration, invasion, proliferation and apoptosis, as well as the changes in skeleton and morphology of medulloblastoma. To testified the supposed functions deeply in mutiple point of views, Above all, our purposes were to understand the biological function of the protein encoded by Racl and to elucidate its mechanism in the development of tumor. This mechanism was the basis of the comprehensive function study of Racl, and provided a theoretical basis for the gene therapy of medulloblastoma.Methods1. Racl expressed in the gliomas, medulloblastomas and its clinical significanceThe collection of human wax block of glioma, medulloblastoma and normal brain tissue, Racl protein was detected by immunohistochemical methods in these tissues. To collect fresh human glioma, medulloblastoma and normal brain tissues respectively, the mRNA expression and Racl protein were detected using RT-PCR and Western blot methods in above-mentioned tissuses.2. The relationship of Racl and invasion and metastasis in medulloblastoma cells(Daoy cells)Gene recombination techniques were used to construct eukaryotic expression vector of Rac1 shRNA, then Rac1shRNA were tranfected into Daoy cell to find possible mechanism of tumor occurrence, migration and invasion. First, Rac1 shRNA were transfected into Daoy cell, then tumor morphology and cytoskeleton changes were obeserved in Daoy cell line after Rac1 gene silenced and enhanced respectively.2.1 Construction of Eukaryotic expression vector of Racl shRNA Nucleotide sequence of Rac1 was planned to cloned in the vector of pGPU6/Neo, recombination vector were enzymed and sequenced.2.2 Transient transfection of Racl-shRNA was carried out using Lipofectamine 2000. After 48 hours, we had used 800mg/L G418 sulfate to select the transfected cells and pick the monoclonal cells. At last we obtained the stably transfected cell.2.3 To detect the mRNA and the protein of Rac1 by RT-PCR and western blotting separately.2.4 To observe the influence of Racl on the morphology and cytoskeleton of Daoy cells by confocal microscope, including the changes of Racl, such as making the Rac1 gene silence or increase.2.5 Colony-forming cell assays was to investigate the influence of the Daoy's proliferation.2.6 Cell monolayer scratch was to analysis the influence of the Racl on the Daoy's migration.2.7 Boyden chamber was to examine the ability of Daoy's invasion after silence of Rac1 gene.2.8 Relation of Racl and cell cycle and apoptosis in medulloblastoma cells Daoy cells were separately transfected with pGPU6/Neo-Rac1, Rac1-shRNA and pGPU6 expression vector plasmid, and then the apoptosis of three group's Daoy cells were detected by flow cytometry with Annexin V-FITC and PI staining method. Then, the influence of silence of Racl on cycle and apoptosis in Daoy cells was studied.The Results1 Rac1 protein were detected in medulloblastoma, astrocytoma and normal brain tissue respectively.Racl immunohistochemical positive cells were cytoplasmic and membrane staining. Racl positive expression show in varying degrees in 112 cases of astrocytic tumors, range of Racl LI (Racl Label inedex) was of 2.3%～63.0%, an average of 13.80% for Racl LI, the comparison among WHOⅠ～Ⅳastrocytic tumors had statistically significant difference (P<0.01). Racl expression is closely related to the tumor degree (r= 0.484, p<0.01), and it had no correlation with the patient's age and sex. Racl positive expression show in varying degrees in 40 cases of medulloblastoma tissues. Rac1 LI was 23.4%～87.5%, with an average 50.5% for the Racl LI, seldom positive expressions were found in normal brain tissues. Racl mRNA and Racl protein were highly expressed in fresh tissues of astrocytic tumors and medulloblastoma tumor, and did not in normal brain tissues. The expression of the above tissues, the comparison between astrocytic tumors tissues and normal brain tissues had statistically significant difference P<0.01. medulloblastoma tumor fresh tissues compared with normal brain tissues, there is statistically significant, P<0.01;2. Construction and identification of recombinant vector Rac1RNAi2.1 Racl shRNA eukaryotic expression vectors were built Successfully, and sequencing of insert sequence were confirmed the same as the designed one exactly through digestion and and sequence.2.2 Daoy cells transfected recombinant plasmid with resistance to G418 filtered stabilized cell-clone;2.3 After established Racl knockdown in Daoy cells, RT-PCR and Western blotting were carried out to testified that endogenous Racl mRNA and protein levels were suppressed by Rac1 shRNA.2.4 Phenotypic changes of cytoskeleton and membrane pseudopod in Daoy cells after Rac1 gene silence: After transient transfection of Rac1 shRNA, both F-actin stress fibers formation and cross-linked actin network were significantly reduced compared to the vector-control. Lamellipodia and pseudopodia formation were also inhibited in Racl-transfected Daoy cells. Another striking feature in response to transfection Rac1-shRNA was the reduction of actin-positive membrane ruffles.2.5 By plate cloning experiments, the proliferation of Daoy cells were inhibited significantly after transfected with pGPU6/Neo/Racl-312.2.6 We also determined the effect of Racl knockdown on tumor cell migration capacities. We found that Daoy cell migration was dramatically reduced, i.e., after wound scratch, only 90±10 of the tumor cells that were transfected with Racl shRNA vector migrated into the wound area compared with 74±11 of the dominant-negative Rac1N17-transfected tumor cells and 276±11 of the control vector-transfected tumor cells. In contrast, transfection of constitutively active Rac1L61 vector resulted in 216±12 of the closed wound after 24 h. Quantitatively, Daoy cells transfected with Racl-shRNA following wound scratch significantly inhibited the wound healing compared with the control cells at each of the time points.Transwell matrigel invasion assay was performed to assess the role of Racl knockdown in suppression of tumor cell invasion capacity. Knockdown of Racl expression by shRNA inhibited tumor cell invasion through Matrigel-coated membranes in Daoy cells compared with vector-only control. In contrast, the constitutively active Rac1L61 transfection increased the invasion capacity of Daoy cells. The ratio of the invaded cell numbers with Rac1 shRNA, Rac1N17, Rac1L61 and Control group was 44±5,35±5,90±5, and 76±7, respectively.2.7 Daoy cells transfected with Racl-shRNA plasmid after 48 hours detected by flow cytometry showed that cell cycle was delayed in the G0-G1 phase and the proportion of G0-G1 phase cells increased significantly to 80.93%, while the proportion of S period cells were reduced to 11.86%. The proportion of G0-G1 phase and S phase cells in plasmid control group (control) were 58.80% and 30.67% repectively. The cell cycle of Daoy was inhibited by deletion of Racl (P＜0.05).2.8 The percentage of apoptotic cells were measured by flow cytometry. Apoptosis rate of Daoy cells transfected with Racl-shRNA plasmid was 36.767±3.950%, while apoptosis rate of plasmid control group was 8.567±0.987%, and deletion of Rac1 increased the cell apoptosis in Daoy cells (p<0.01).Conclusions:Sum up, Racl, high expression especially in the medulloblastoma indicates that Racl played a positively regulating role in the evolution course of medulloblastoma, involved in controling of proliferation, clonality, formation of tumor cell cytoskeleton, mobility and invasion of medulloblastoma, on the other hand, Racl played a negative role in the course of apoptosis. As a newly found cancer gene, our data firstly demonstrated that Racl has the potential to be the molecular target in the treatment of medulloblastoma.