恶性胶质瘤miR-29s表达异常减少在其发生发展中的作用及分子机制研究

Primary glioblastoma (pGB) is the most common malignant gliomas. The reduced protein level of wild type tumor suppressor p53 (wtp53) is the crucial cause of tumorigenesis in pGB. Recent studies indicated that the CDC42/PAKs signaling pathway could promote the ubiquitination of wtp53 and accelerate its degradation. Our previous work showed that CDC42 mRNA is a target of miR-29a/b/c. The expression level of the miR-29c in the glioma groups were significantly lower than the control and were related with the malignancy grades of glioma, but almost not in the pGB. Introduction of miR-29a/b/c in glioblastoma cell lines could significantly upregulate wtp53 levels and induce apoptosis. According to these results, we propose the hypothesis that down-regulation of miR-29a/b/c expression in glioblastoma cells could remove the post-transcriptional inhibition of target gene CDC42 and then enhance the activity of PAKs kinase. And this process could lead to wtp53 protein degradation and finaly promote tumorigenesis. The following parts are the central idea of this project: ① clearing the effects of the expression level of miR-29a/b/c on tumorigenesis of glioblastoma and assessing whether they can be used as evaluation index of grading and prognosis in gliomas; ② revealing the molecular mechanisms of aberrant reduction of wtp53 protein in pGB and discussing the roles of CDC42/PAKs in the regulatory process; ③ clarifying the roles of aberrant miR-29s/CDC42/PAKs/p53 pathway in the tumorigenesis of glioblastoma. Through the research of this subject, we try to provide additional proof for the molecular mechanisms for tumorigenesis of glioblastoma, and we also hope we could provide more theoretical evidence for developing new technology of the diagnosis and treatment of glioblastoma.

恶性胶质瘤中最常见的是原发性胶质母细胞瘤(pGB), 野生型p53蛋白(wtp53)减少与其发生发展密切相关; 近来发现CDC42/PAKs通路可促进wtp53泛素化加速其降解。我们已证实CDC42是miR-29s的靶基因, miR-29c表达与胶质瘤级别呈负相关, 上调miR-29s可显著增加pGB细胞wtp53水平; 高度提示miR-29s下调可能是导致pGB细胞CDC42/PAKs通路过度活化及wtp53减少的重要原因。本项目的中心思想是：①确定miR-29s表达变化与胶质瘤发生发展的关系及其对评估肿瘤分级和患者预后的实用价值; ②探讨pGB细胞miR-29s下调导致wtp53减少的确切机制及CDC42/PAKs在其中的作用; ③揭示miR-29s/CDC42/PAKs/p53通路异常在恶性胶质瘤发生发展中的作用。旨在为阐明其发病机制提供新线索, 为研发诊疗新技术提供客观参考依据。

胶质瘤是最常见的颅内原发性肿瘤，目前对胶质瘤发生发展的确切分子机制尚缺乏深入了解，严重制约了对胶质瘤有效治疗策略的研究。越来越多的证据表明，分子靶向治疗与基因干预技术有可能成为未来治疗胶质瘤的有效手段。而阐明胶质瘤发生发展的分子机制，找出与其发生发展密切相关的关键责任基因和责任调控通路，是优选高效特异性治疗靶点与靶通路亟待解决的关键问题。.本项目的研究方向是明确miR-29a/b/c在胶质瘤组织中的表达变化，及其与胶质瘤良恶性级别、肿瘤细胞生物学特征及患者预后的关系，探讨miR-29a/b/c在胶质瘤发生发展中的重要作用及其具体分子机制，旨在为阐明胶质瘤发生发展的分子机制提供重要的线索，为基因治疗靶点优选提供客观参考依据。.本项目研究以大宗人胶质瘤组织标本和胶质母细胞瘤细胞系为研究对象，采用一系列组织病理学、细胞生物学及分子生物学等研究方法，取得的重要研究结果具体如下：① miR-29a/b/c是胶质瘤的重要抑瘤miRNAs，其在胶质瘤组织中表达异常减少且随胶质瘤良恶性级别升高而降低，可作为评价胶质瘤良恶性级别及患者预后的重要参考指标；② 人胶质瘤组织中普遍有CDC42、TRAF4过表达并随胶质瘤良恶性程度的升高而增加，可作为评价胶质瘤良恶性级别及患者预后的参考指标，miR-29a/b/c表达异常减少是胶质瘤组织CDC42、TRAF4过表达的重要原因；③ miR-29a/b/c通过直接靶向敲低CDC42表达，阻断胶质瘤细胞AKT和MDM2的磷酸化级联激活而诱导的p53泛素化降解，从而促进胶质瘤细胞p53蛋白蓄积，调控p53介导的细胞凋亡。④ miR-29a/b/c可通过直接靶向敲低CDC42表达抑制胶质瘤细胞PAK和LIMK的磷酸化级联激活，抑制cofilin磷酸化失活，从而抑制F-actin的聚合，抑制胶质瘤细胞迁移侵袭能力。⑤ miR-29a/b/c可通过直接靶向敲低TRAF4表达，阻断胶质瘤细胞AKT磷酸化激活而导致的c-Myc和cyclin D1蛋白表达上调，抑制胶质瘤细胞由G1期进入S期而抑制胶质瘤细胞的增殖。以上研究结果为进一步研发胶质瘤分子靶向治疗新技术及新药物提供了重要的新线索和客观的参考依据，对提高胶质瘤诊疗水平起到积极的促进作用。