MiR-5096 通过激活星形胶质细胞促进胶质瘤侵袭的机制研究

The invasiveness of glioma is the primary reason for poor survival following treatment. Interaction between glioma cells and surrounding astrocytes are crucial to invasion. Our previous work reveal that glioma invasion is promoted by reactive astrocytes when miR-5096 are transferred from glioma cells to neighboring astrocytes. However, the downstream effect of miR-5096 is still unknown. Bioinformatics’ analysis indicate that miR-5096 are involved in gap junction, paracrine and exosome pathways which are also highly related in glioma invasion. In this project, we propose to determine the molecular mechanisms by which miR-5096 activate astrocyte and promote glioma invasion by modulating the above pathways. Using report gene technique we will seek to identify the protein target of miR-5096. Moreover, we will further validate those finding in vivo by using a mouse glioma model. Our findings from this proposal will offer new insights about the modulating effect of miR-5096 on glioma microenvironment, and provide a proof-of-principle in developing a novel application in miRNA-based therapy in glioma control.

胶质瘤侵袭性生长的特性是其复发率高预后差的关键。研究发现胶质瘤周边的星形胶质细胞被激活而促进胶质瘤的侵袭但机制不明。我们的前期工作证实星形胶质细胞的激活及促侵袭作用与胶质瘤细胞中miR-5096向星形胶质细胞传递相关，但miR-5096的下游机制仍待探索。生物信息学分析提示miR-5096可能调控细胞缝隙连接、旁分泌通路及外泌体等细胞间相互作用途径。本项目将首先在miR-5096激活的星形胶质细胞中分别检测及改变此三条通路的功能，观察对胶质瘤侵袭能力的影响，从而分析miR-5096激活星形胶质细胞后调控胶质瘤侵袭的内在机制，并运用报告基因等技术鉴定受miR-5096调控的靶蛋白。进一步采用小鼠胶质瘤模型，结合3D透明大脑成像技术在动物体内验证上述机理。研究力争揭示miR-5096对胶质瘤微环境的调控机制，为控制胶质瘤侵袭提供分子靶标和理论依据。

胶质瘤侵袭性生长的特性及对化疗药物的耐药性是其复发率高预后差的关键。microRNA是一类广泛分布的非编码小RNA，能通过靶向作用于mRNA, 导致翻译抑制以及RNA 降解来调节基因表达。本项目对miRNA在胶质瘤的侵袭性及耐药性的形成过程所发挥的作用进行探索。我们首先检测了miR-5096对胶质瘤细胞的增殖和迁移能力的影响，结果显示过表达miR-5096或使用miR-5096的抑制剂均不影响胶质瘤的增殖和迁移。细胞水平上检测miR-5096激活星形胶质细胞而促胶质瘤侵袭的机制，我们在星形胶质细胞上过表达miR-5096,观察miR-5096激活的星形胶质细胞的上清液是否促进胶质瘤的侵袭，结果显示该上清液对胶质瘤细胞的侵袭能力并无明显影响。由此我们证实miR-5096激活星形胶质细胞而促胶质瘤侵袭作用并不通过上清途径，亦排除外泌体及细胞因子途径。我们进一步对miRNA是否通过细胞缝隙连接途径影响胶质瘤恶性表型进行探索。qPCR及Western blot 验证miR-5096对Cx43蛋白无影响，但我们同时发现另一条miRNA, miR-23b-3p能够明显下调胶质瘤细胞中Cx43的表达，并且miR-23b-3p能显著影胶质瘤细胞对化疗药物替莫唑胺（temozolomide, TMZ）的敏感性。为此我们增加了对miR-23b-3p影响胶质瘤TMZ敏感性的机制探索，细胞实验和动物体内证实miR-23b-3p调控TMZ耐药，其靶标蛋白为Cx43。本研究揭示了miRNA对胶质瘤侵袭及耐药的调控机制，为控制胶质瘤恶性表型提供分子靶标和理论依据。