脑胶质瘤中Hedgehog通路介导的长链非编码RNA-MEG3作用机制的研究

Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults. Patients with newly diagnosed glioblastoma have a median survival of approximately 1 year which mainly resulted from its poor responses to all therapeutic modalities .Our previous studies showed that aberrant activation of Hedgehog signaling pathway has been observed in gliomas and inhibition of the pathway’s activity by drugs or miRNAs affected the biological behaviors of glioma cells and the self-renewal of tumor stem cells. After we used RNA interference technology mediated by lentivirus vectors blocking the hedgehog signaling activity, we found that according to the expression profile ,the long non-coding RNA-MEG3 upregulated. Meanwhile, We profiled the lncRNA expression signatures in 220 gliomas and found that expression of MEG3 was negatively correlated with glioma grade and poor prognosis..Based on the above results of the gene expression profiles,we aimed to verify the expression of MEG3 in gliomas specimens and analysed the correlation between MEG3 and GLI1 genes. Subsequently, we intended to investigate the negative role of GLI1 on MEG3 mediated by DNMT1. We plan to identify the mechanism of MEG3 in gliomas,which may modulate the activity of P53 pathway and its downstream target genes ( p21,GDF15) through activation of P53. Moreover, we would also like to investigate the gliomas biological behaviors including proliferation, apoptosis, stemness and the sensitivity to TMZ after modulation of MEG3 in vitro and vivo. Finally, we aimed to explore the effect of P53 on GLI1’s expression and trans- criptional activity..The present study may provide insides for understanding the comprehensive networks of MEG3/P53/GLI1 on glioma progression. Morever, it will provide a new treatment strategy in gliomas.

课题组前期工作发现，利用慢病毒介导的siRNA技术敲低核转录因子GLI1的表达谱结果显示，长链非编码RNA-MEG3表达显著上调；进一步对220例胶质瘤患者临床资料和组织样本基因表达谱分析发现，MEG3在胶质瘤中呈低表达，与级别呈负相关。.本项目在上述基础上，验证胶质瘤中MEG3表达、与肿瘤级别、预后的关系及敲低GLI1后对MEG3表达的影响；研究GLI1作用DNMT1对MEG3负向调控作用；探索MEG3转录调控P53的作用机制及对p53通路活性及下游靶基因（p21、GDF15）的影响；研究不同MEG3表达在体内外对胶质瘤增殖、凋亡、干细胞“干性”及TMZ敏感性的作用；探讨P53对GLI1表达及转录活性影响，解析MEG3/P53/GLI1调控环路在临床胶质瘤早期诊断及预后判断中的意义；.本课题可揭示Hedgehog通路介导下MEG3发挥作用的分子机制，为胶质瘤的靶向治疗提供新的理论依据。

本项目已按计划完成所有研究内容，取得创新型研究成果，其中研究内容主要集中在以下几个方面：课题组发现利用慢病毒介导的RNA干扰技术敲低Hedgehog通路核转录因子GLI1后进行表达谱的分析，发现DNMT1表达下调，lncRNA-MEG3表达水平显著上调，差异具有明显统计学意义；同时，进一步对220例胶质瘤患者的组织样本的基因表达谱研究发现，GLI1与DNMT1表达呈正相关；DNMT1可以影响MEG3启动子区甲基化水平，进而调控MEG3表达水平通过调控p53信号通路负反馈作用Hedgehog通路；相比于级别胶质瘤，高级别胶质瘤中MEG3表达水平显著下调；单就GBM患者而言，MEG3高表达的患者的总生存期要优于MEG3低表达患者，二者具有显著的统计学差异。另外，课题组还进一步发现miR-326联合姜黄素通过Hedgehog信号通路强化对胶质瘤细胞的抑制作用，在体内实验中显著延长模型鼠生存期。利用微阵列及生物信息学分析表明，DUSP5是p68基因的一个靶点，生物学实验进一步证实p68基因负性调节DUSP5，稳定上调DUSP5后显著地抑制胶质瘤细胞的增殖、侵袭以及迁移能力，反之亦然；与此同时，上调DUSP5后能抑制ERK的磷酸化，提示这一机制可能是通过抑制ERK信号通路活化作用的。同时还进一步对胶质瘤细胞的转移及上皮向间质转化等问题研究，研究结果表明，Notch1在胶质瘤中表达上调，且随着级别升高表达强度不断增加；敲低Notch1后能明显抑制胶质瘤细胞的侵袭及转移能力；同时还发现Notch1是小RNA miR-139-5p的一个靶点，深入的研究表明miR-139-5p可以通过调控Notch1影响肿瘤细胞的上皮向间质转化；体内外的实验均证实该研究的有效性，这也为胶质瘤的治疗提供一个新的思路。在本项目资助下，发表SCI收录论著3篇，国家级核心期刊1篇，最高影响因子为4.372，并有多篇在投。同时，以本项目研究为中心，我课题组扩充脑胶质瘤数字化生物样本库，收集、扩充并检测胶质瘤肿瘤样本，构建原代肿瘤细胞系，为后续的实验研究提供稳定的平台。