Cath B and muPAR siRNA constructs regressed glioma growth

描述（由申请人提供）：恶性脑肿瘤是最难治疗的癌症之一，并且仍然是无法治愈的。胶质瘤是最常见的脑肿瘤类型，有不同的级别，患者的预后与级别成反比。我实验室的长期目标是了解人类胶质瘤侵袭性的细胞和分子机制。我的实验室一直活跃于蛋白酶和脑肿瘤生物学的研究，目前得到的数据表明蛋白酶的变化与肿瘤分级的变化是相关的。假设：1)通过表达组织蛋白酶B和uPAR信息的siRNA载体调控人胶质瘤组织蛋白酶B和uPAR的表达可抑制肿瘤生长、侵袭和血管生成；2)在胶质瘤细胞系中通过双链RNAi构建调控组织蛋白酶B和uPAR蛋白水平可降低参与细胞存活、迁移、增殖和血管生成的信号通路分子的水平。解决这些假设的具体目标如下：评价siRNA靶向组织蛋白酶B和uPAR （pCU/AdCU）载体对胶质瘤细胞体外生长、附着、迁移和侵袭的影响。测定表达siRNA抗组织蛋白酶B和uPAR （pCU/AdCU）载体对胶质母细胞瘤细胞系组织蛋白酶B和uPAR水平的影响。Ib.比较pCU/AdCU构建物对胶质瘤生长、粘附、凋亡和迁移的影响与对照/模拟EV（空载体）和SV（重组载体）的体外比较。c.在对照/模拟、空载体EV和乱载体SV体外模型中，研究pCU/AdCU构建体对人胶质母细胞瘤细胞侵袭行为的影响。具体目标2。测定pCU/AdCU在体内对裸鼠侵袭性和致瘤性的影响。2 a。研究pCU/AdCU构建物对裸鼠颅内注射的人胶质母细胞瘤细胞系（SNB19和U251）颅内肿瘤生长或侵袭性的影响。2 b。确定pCU/AdCU构建体对两种异种移植裸鼠颅内肿瘤生长或侵袭性的影响。具体目标3。探讨pCU/AdCU对细胞增殖、凋亡和血管生成的分子机制的影响。3 a。评价pCU/AdCU对细胞增殖分子机制的影响。3 b。评价pCU/AdCU对细胞凋亡分子机制的影响。3 c。评价pCU/AdCU对体外和体内模型脑血管生成的影响。我们预计这些结果将大大增加我们对组织蛋白酶B和uPAR分子如何调节的理解；因此，获得的信息应该有助于开发治疗胶质母细胞瘤的新治疗方法。

DESCRIPTION (provided by applicant): Malignant brain tumors represent one of the most refractory cancers to therapy and remain incurable. Gliomas represent the most common type of brain tumors that occur in various grades, with the patient's prognosis inversely proportional to the grade. The long-term objective of my laboratory is to understand the cellular and molecular mechanisms that underlie tumor invasiveness in human gliomas. My laboratory has been active in the study of proteases and the biology of brain tumors, and data generated so far have indicated that changes in proteases are correlated with the changes in the grade of the tumors. The hypotheses are: 1) Regulation of cathepsin B and uPAR expression in human gliomas via a vector expressing siRNA for cathepsin B and uPAR message will inhibit tumor growth, invasion and angiogenesis and 2) Regulation of cathepsin B and uPAR protein levels by bicistronic RNAi constructs in glioma cell lines decrease the level of signaling pathway molecules which are involved in cell survival, migration, proliferation and angiogenesis. The Specific Aims to address these hypotheses are as follows: Specific Aim 1. Evaluate the effect of vectors expressing siRNA targeting cathepsin B and uPAR (pCU/AdCU) on glioma cell growth, attachment, migration and invasion in vitro, la. Determine the effect of vectors expressing siRNA against cathepsin B and uPAR (pCU/AdCU) on cathepsin B and uPAR levels in glioblastoma cell lines. Ib. Compare the effect of the pCU/AdCU constructs on glioma growth, adhesion, apoptosis and migration with that of control/mock EV (empty vector) and SV (scrambled vector) in vitro. Ic. Investigate the effect of the pCU/AdCU constructs on the invasive behavior of human glioblastoma cells in in vitro models with that of control/mock, EV (empty vector) and SV (scrambled vector). Specific Aim 2. Determine the in vivo effects of pCU/AdCU on invasion and tumorigenicity in nude mice. 2a. Access the ability of the pCU/AdCU constructs in pre-established intracranial tumor growth or invasiveness of human glioblastoma cell lines (SNB19 and U251) injected intracranially in nude mice. 2b. Determine the effect of pCU/AdCU constructs in pre-established intracranial tumor growth or invasiveness of two xenografts in nude mice. Specific Aim 3. Determine the effect of pCU/AdCU on the molecular mechanism of proliferation, apoptosis and angiogenesis. 3a. Evaluate the effect of pCU/AdCU on the molecular mechanism of proliferation. 3b. Evaluate the effect of pCU/AdCU on the molecular mechanism of apoptosis. 3c. Evaluate the effect of pCU/AdCU on cerebral angiogenesis in both in vitro and in vivo models. We anticipate that these results will substantially augment our understanding of how cathepsin B and uPAR molecules are regulated; thus, information gained should be of help in developing new therapeutic approaches to treat glioblastomas.