RND3对Snail1信号通路的作用及与人脑胶质母细胞瘤侵袭的关系

This proposal objects to investigate the regulation of RND3 (a small Rho GTPase) on Snail1 signaling and effect of RND3 on glioblastoma multiforme（GBM）invasion and associated molecular mechanism. RND3 is a endogenous inhibitor of Rho kinases, and can inhibit GBM cells invasion, however, the mechanism was unclear. studies have reported that RND3 regulates E-cadherin, whcih is the target gene of Snail1, in hepatocellular carcinoma. our previous study found that RND3 can bind Snail1 and decreased Snail1 protein expression level in GBM cells. As we known, Snail1 is critical for Snail signaling which is one of the most important signaling in GBM invasion. So we hypothesize that RND3 inhibits GBM cells invasion through inhibiting Snail1 signaling. We will detect the effect and mechanism of RND3 in regulating Snail1 signaling in human GBM samples, GBM cells and RND3 nude mice, we also will detect whether RND3 regulates Snail1 signaling is the critical mechanism of RND3 inhibiting GBM invasion. We believe that our work would strengthen the understanding of GBM mechanism,enrich experimental evidence for clinic GBM therapy and provide new molecular targeted for GBM gene therapy.

本项目重点研究小G蛋白RND3对Snail1信号通路的作用及与脑胶质母细胞瘤（GBM）侵袭的关系。RND3是Rho激酶抑制因子，可以抑制GBM的侵袭，但其分子机制尚不清楚。文献报道，RND3在肝癌中可以调节Snail1信号通路下游基因E-cadherin，而我们前期研究发现，RND3与Snail1蛋白结合，并降低Snail1蛋白表达量，Snail1蛋白是Snail1信号通路的关键蛋白，Snail1信号通路是调控GBM侵袭的关键信号通路。因此，本项目提出假说：RND3通过抑制Snail1信号通路，抑制GBM侵袭。本项目将从人脑GBM标本、胶质母细胞瘤细胞、基因敲除小鼠水平探讨RND3对Snail1信号通路的调控作用、具体分子机制以及RND3是否通过调控Snail1信号通路调控GBM侵袭。本项目有望增加对GBM侵袭分子机制的进一步理解，为GBM的治疗提供新靶点，并为以后的临床应用提供实验依据。

研究背景：.脑胶质瘤是中枢神经系统最常见最致命的原发性恶性肿瘤，严重威胁患者生命健康。如何寻找有效的治疗方法、控制脑胶质瘤的侵袭性生长和术后复发，仍是临床和基础医学研究的一大难题。尤其是胶质母细胞瘤（glioblastoma multiforme, GBM），在胶质瘤中所占比例高达50%，即便是术后联合应用放化疗，效果仍然不理想，患者的中位生存时间只有12-15个月。GBM治疗效果不佳的主要原因之一是GBM的高侵袭性，其侵入正常脑组织或者脊髓组织，导致手术无法全部切除肿瘤，并最终导致复发。遗憾的是，关于GBM侵袭的机制目前尚不清楚，而GBM治疗效果的提高依赖于对其分子机制的进一步研究。..主要研究内容： .本项目重点围绕小G蛋白RND3（Rho family GTPase 3, RND3，又称RhoE)对Snail1信号通路的作用及与GBM侵袭的关系进行研究。..重要结果：.（1）.在人脑GBM标本中，RND3与 E-cadherin、Claudin的mRNA及蛋白表达量呈正相关；.（2）.体外实验结果显示，在U251细胞内，过表达RND3，GBM细胞侵袭性降低，低表达RND3，GBM细胞侵袭性增加；.（3）.体内实验结果显示，过表达RND3的稳定细胞在裸鼠内形成的肿瘤更小，肿瘤侵袭能力更弱；.（4）.在GBM细胞内，过表达RND3，E-cadherin和Claudin的mRNA和蛋白表达量增高，低表达RND3, E-cadherin和Claudin的mRNA和蛋白表达量降低；.（5）.在RND3基因敲除小鼠的脑组织内，E-cadherin和Claudin的mRNA和蛋白表达量降低；.（6）.在GBM细胞及人脑GBM组织中，过表达RND3，Snail1蛋白表达量降低，低表达RND3，Snail1蛋白表达量升高，但Snail1的mRNA表达量无明显改变；在RND3基因敲除小鼠的脑组织内，Snail1蛋白表达量升高；.（7）.RND3与Snail1直接结合，促进其泛素化，降低Snail1蛋白表达量，进而抑制Snail1信号通路。..重要意义.实验结果将有助于了解GBM细胞异常侵袭的机理，指导早期临床诊断。同时本课题在国内外首次研究人脑GBM中RND3对Snail1信号通路的调控规律，对于揭示GBM发病及恶化的分子机制，寻求GBM治疗新方法具有重要意义。