EGFR突变型NSCLC通过外泌体促进脑星形胶质细胞VEGF分泌的机制研究

The incidence of brain metastases was higher in patients with EGFR positive mutation than those with wild type. Changing the permeability of blood-brain barrier (BBB) was the key step in tumor brain metastasis. Increasing the secretion of VEGF in brain astrocytes could change the permeability of BBB. In our previous study, we found that the exosomes derived from the EGFR-mutated NSCLC cell lines could be internalized by astrocytes via endocytosis and was correlated with brain metastases. Further, we found that the expression of miR-146a-5p was significantly higher in exosomes derived from EGFR-mutated NSCLC cell lines and in brain tissues of the corresponding tumor bearing mice. Bioinformatic analysis found SMAD4 and NF2 were the target genes of miR-146a-5p, which were related to the secretion of VEGF. We hypothesized that miR-146a-5p in EGFR mutant positive NSCLC exosomes could promote the secretion of VEGF in brain astrocytes via SMAD4/VEGF and/or NF2/PAK1/VEGF pathway. In this study, we will use multiple techniques and models including, RNA sequencing, knock-down and overexpression of miRNA, tumor bearing mice models to elucidate that EGFR mutant positive NSCLC could increase the secretion of VEGF in brain astrocytes via the exosomes. Ultimately, we determine to provide a theoretical basis for the occurrence of brain metastases in EGFR mutant NSCLC.

EGFR突变型NSCLC易发生脑转移。血脑屏障通透性改变是肿瘤脑转移的关键环节，脑星形胶质细胞VEGF分泌增多在此过程中发挥重要作用。项目组前期发现：NSCLC细胞株外泌体可被脑星形胶质细胞吞噬，并与脑转移相关；EGFR突变型NSCLC细胞株外泌体及相应荷瘤鼠脑组织miR-146a-5p表达显著高于野生型；生信预测提示SMAD4和NF2为miR-146a-5p潜在靶基因，两者可影响VEGF分泌。我们提出假说：EGFR突变型NSCLC通过外泌体miR-146a-5p靶向脑星形胶质细胞SMAD4和/或NF2，活化SMAD4/VEGF和/或NF2/PAK1/VEGF通路，促进VEGF分泌。本项目拟通过RNA测序、敲减和过表达miRNA、动物模型建立等方法，阐明EGFR突变型NSCLC通过外泌体促进脑星形胶质细胞VEGF分泌的分子机制，并为EGFR突变型NSCLC脑转移发生提供一定的理论基础。

表皮生长因子受体（epithelial growth factor receptor，EGFR）突变非小细胞肺癌患者脑转移发生率高，进一步明确相关机制对于预防或干预脑转移的发生有重要意义。血脑屏障通透性的增加在肿瘤脑转移中起重要作用，而脑星型胶质细胞是血脑屏障的重要构成部分，其分泌的VEGF对于改变血脑屏障通透性有重要作用。本项目聚焦于探索EGFR突变非小细胞肺癌易于脑转移的相关机制，探索肿瘤细胞是否通过外泌体远程影响脑星型胶质细胞VEGF的分泌。研究通过体外与体内实验证实了肿瘤细胞来源的外泌体能够被星型胶质细胞摄取，同时能够改变星型胶质细胞VEGF的分泌水平。体外研究结果提示EGFR突变细胞来源的外泌体促进星型胶质细胞VEGF分泌的程度明显高于EGFR野生型细胞。裸鼠体内实验发现与EGFR突变细胞系来源的外泌体同期注射能够明显增加EGFR野生型细胞脑转移的发生率。以上体内外实验证实EGFR突变细胞分泌的外泌体能够促进肿瘤细胞的脑转移。随后的外泌体microRNA测序及qRT-PCR的验证发现，来自于EGFR突变细胞系的外泌体中miR-146a-5p表达量明显升高，同时生信研究发现与VEGF表达相关的基因SMAD4和NF2是miR-146a-5p潜在的靶点，表明EGFR突变细胞可能是通过外泌体运输miR-146a-5p进入脑星型胶质细胞，进而改变星型胶质细胞中SMAD4和NF2的表达，改变其VEGF的分泌。