Nrf2/miR-29b/Sp1正反馈环路影响胶质瘤化疗敏感性的机制研究

The prognosis of glioma is severely affected by chemoresistance. But the mechanism of glioma chemoresistance still needs further research. It has been proved that Nrf2 regulates proliferation, apoptosis, migration, angiogenesis and differentiation of glioma by our previous study. And the results of preliminary experiment indicate that Nrf2 regulates the chemoresistance in glioma. miR-29b has been proved to be an possible Nrf2-target miRNA by ChIP-seq analysis. In view of the existence of miR-29b/Sp1 regulatory loop and the expression of Nrf2 regulated by Sp1, the positive feedback loop of Nrf2/miR-29b/Sp1 may exit, which would persistently activate the Nrf2-ARE signal pathway and improve the malignant phenotype. So the hypothesis is that Nrf2/miR-29b/Sp1 positive feedback loop may involve in glioma chemoresistance. Based on preliminary study, the role of Nrf2 in chemoresistance and the relationship of Nrf2, miR-29b and Sp1 in glioma would be further studied by molecular biological, immunostain and transgenes technique in vivo and in vitro. Also the role of miR-29b in glioma chemoreisistance would be investigated. This study is source of innovation. And the results will reveal the mechanism of glioma chemoresistance, which will provide new thread for effective therapy and new target for drug development.

化疗耐药严重影响胶质瘤患者的预后，但胶质瘤化疗耐药的确切机制尚不清楚。课题组前期研究已首次证实：Nrf2可调节胶质瘤的增殖、凋亡、迁移、血管生成及分化，预实验结果提示Nrf2可调控胶质瘤化疗敏感性。基于1)miR29b可能受Nrf2调控；2)miR-29b/Sp1环路存在；3)Sp1可调控Nrf2表达，推测可能存在Nrf2/miR-29b/Sp1正反馈环路，持续激动ARE基因从而导致肿瘤恶性表型增加，并提出“Nrf2/miR-29b/Sp1正反馈环路影响胶质瘤化疗敏感性”的假说。本项目拟在前期研究基础上，通过体内外实验及分子生物学、转基因、miRNA等技术，进一步验证Nrf2水平与胶质瘤耐药的关系，检测Nrf2、miR-29b、Sp1在胶质瘤内表达的相互影响及其与化疗的关系，探讨miR-29b能否调控胶质瘤化疗耐药。本研究属于源头性创新，将有利于寻找改进胶质瘤化疗效果的可行方法。

化疗耐药严重影响胶质瘤患者的预后，但胶质瘤化疗耐药的确切机制尚不清楚。课题组结合前期研究结果及文献复习，提出“Nrf2/miR-29b/Sp1正反馈环路影响胶质瘤化疗敏感性”的假说并进行验证。利用人胶质瘤标本，免疫组化检测复发前后胶质瘤内Nrf2表达水平改变。利用胶质瘤细胞系，多种化疗药物不同时间点、不同浓度检测Nrf2-ARE通路表达改变，并在体外、体内检测Nrf2表达下调后TMZ处理后胶质瘤凋亡、焦亡的改变。筛选U251TMZ耐药细胞系，检测耐药前后Nrf2-ARE通路改变情况。随后利用Nrf2、Sp1过表达质粒及miR-29b inhibitor分别转染U251、A172，检测转染后三者表达水平的改变，验证三者表达水平是否存在正反馈环路。最后利用miR-29b mimic转染，提高胶质瘤内miR-29b水平，体内、体外检测其对TMZ处理后胶质瘤凋亡、焦亡的影响。结果发现不同化疗药物刺激后，胶质瘤U251、A172细胞内Nrf2-ARE通路出现激活，复发胶质瘤标本与原发标本相比，Nrf2表达明显增多。而将胶质瘤细胞内Nrf2表达抑制后，TMZ作用下发现存活率下降，平板克隆形成率也明显下降。流式检测提示焦亡、凋亡比例增高。裸鼠成瘤实验也证实Nrf2敲除后，TMZ作用下肿瘤瘤体明显缩小。Nrf2、Sp1过表达质粒及miR-29b inhibitor分别转染后发现U251、A172内Nrf2、Sp1表达增高，miR-29b表达下降，这从表达层面证实三者之间存在正反馈调控，可能是胶质瘤内Nrf2持续过表达的重要机制。利用miR-29b mimic转染胶质瘤细胞系提高miR-29b表达水平，导致细胞内Nrf2、Sp1表达水平明显下降，进而导致TMZ作用下平板克隆形成率下降、存活率明显降低。而流式结果也证实mimic转染导致TMZ诱导的焦亡、凋亡比例增高。体内实验也证实mimic瘤内注射导致TMZ作用下肿瘤明显缩小。我们的结果证实在胶质瘤内Nrf2持续高表达是化疗耐药的一个重要机制，而持续高表达的Nrf2可能是Nrf2/miR-29b/Sp1这一正反馈表达环路的结果，利用miR-29b mimic提高miR-29b表达水平，可以抑制该环路内高表达的Nrf2及Sp1，导致细胞耐药性明显改善。