BACH1与FUS相互作用通过非编码RNA调控Hippo通路影响脑胶质瘤干细胞生物学行为的分子机制

BACH1 is highly expressed in brain glioma tissues and GSCs. The down-regulation of BACH1 decreases the proliferation, migration and invasion of GSCs significantly, accompanied by a markedly up-regulated lncRNA-DLX6-AS1 expression. Predicted by JASPAR CORE software, the BACH1 binding sites were found in the promoter region of DLX6-AS1. Co-immunoprecipitation experiment determined the interaction between BACH1 and FUS. Further, the hypothesis is established as follows through a series of our previous work: transcription factor BACH1 is highly expressed in GSCs and might act as an oncogene. The highly expressed BACH1 regulates the expression of DLX6-AS1 by interacting with FUS. The lower expressed DLX6-AS1 might decrease its binding with miR-103-3p to enhance the negative control of miR-103-3p on its target genes WWC3, MST1 and LATS1, and promote YAP to transport into the nucleus. After importing into the nucleus, MEF2B might interact with YAP to regulate the expression of LGALS3 and mTOR positively on one side and control the upstream BACH1 expression positively to form a feedback loop on the other side, which can promote the proliferation, migration, invasion of GSCs and inhibit autophagic cell death. On the basis of our previous work, this project will firstly verify transcription factor BACH1 and RNA binding protein FUS interact with each other and determine their interacting domain. We will further analyze the binding of DLX6-AS1 with miR-103a-3p to find out their binding sites as well as to clarify the effect on biological behaviors of GSCs regulated by DLX6-AS1 via miR-103a-3p. Then we will study the role of miR-103a-3p targeting WWC3, MST1 and LATS1, and find out their binding sites to illustrate the mechanisms in which miR-103a-3p target and control the expression of WWC3, MST1 and LATS1 to regulate the biological behaviors of GSCs. On the basis of above, we will study the interaction between transcription factor MEF2B and YAP and elucidate the mechanisms in which MEF2B interact with YAP to positively regulate the expressions of LGALS3 and mTOR on one side positively regulate upstream BACH1 expression to form a feedback loop on the other side, affecting GSCs proliferation, migration, invasion and autophagic cell death. Finally, we will explore the optimal approach to administer whether with the inhibitor or activator of multiple targets as BACH1, DLX6-AS1 and miR-103a-3p separately or in combination by analyzing the anti-tumor efficacy on glioma in nude mice. This project might not only clarify the effect related to BACH1 on GSCs biological behavior mediated by non-coding RNAs, but also provide a new pathway to enhance the therapeutic efficacy on brain gliomas.

前期发现BACH1在GSCs高表达，沉默后抑制了GSCs的增殖、迁移和侵袭。通过系列前期工作推测，BACH1与FUS互作负性调控DLX6-AS1表达。低表达的DLX6-AS1减少与miR-103a-3p的结合，增强miR-103a-3p对WWC3、MST1和LATS1的负性调控，促进YAP入核。MEF2B与YAP互作通过调控LGALS3和mTOR的表达，以及调控BACH1的表达形成反馈环路，共同促进GSCs的增殖、迁移、侵袭并抑制自噬性死亡。项目研究BACH1与FUS互作及结构域，调控DLX6-AS1表达的机制；研究DLX6-AS1与miR-103a-3p、miR-103a-3p与WWC3、MST1和LATS1的结合作用调节GSCs生物学行为的机制；研究MEF2B与YAP互作后调控LGALS3和mTOR，以及调控BACH1形成反馈环路，影响GSCs增殖、迁移、侵袭及自噬性死亡的作用机制。

本项目深入研究了BACH1与FUS互作通过介导Hippo通路调控胶质瘤干细胞(GSCs)生物学行为的作用与机制。主要研究结果如下：(1)BACH1与FUS在GSCs中的细胞核中存在共定位，BACH1与FUS存在相互作用，BACH1的C-末端结构域是相互作用位点；BACH1与DLX6-AS1存在结合作用，结合位点为-1752～-1738；FUS能够显著增强BACH1和DLX6-AS1的结合能力。(2)BACH1表达沉默通过上调DLX6-AS1，下调miR-103a-3p的表达，增加Hippo通路的核心分子-WWC3、MST1、LATS1和YAP，以及p-MST1、p-LATS1和p-YAP的表达，抑制YAP入核，抑制GSCs的增殖、迁移、侵袭(即抑制GSCs的恶性生物学行为)。(3)在GSCs中，DLX6-AS1低表达，miR-103a-3p高表达。DLX6-AS1过表达通过降低miR-103a-3p，增加WWC3、MST1和LATS1，以及p-MST1、p-LATS1和p-YAP的表达，抑制YAP入核，抑制GSCs的恶性生物学行为。(4)DLX6-AS1过表达显著增加了自噬相关蛋白LC3-II、beclin1等的表达，降低自噬底物p62的表达，促进了GSCs的自噬性死亡。(5)miR-103a-3p分别与WWC3、MST1和LATS1的mRNA的3’UTR区靶向结合。(6)MEF2B与YAP在GSCs的细胞核中存在共定位并互作。(7)MEF2B分别与LGALS3、mTOR和BACH1的启动子区直接结合，促进其转录；MEF2B与YAP的互作一方面正性调控下游LGALS3和mTOR的表达，另一方面正性调控上游BACH1的表达形成反馈环路，共同影响GSCs的增殖、迁移、侵袭以及自噬性死亡。(8)BACH1表达沉默、DLX6-AS1过表达和miR-103a-3p表达沉默单独或联合应用均显著抑制GSCs恶性生物学行为，三者联合应用是抑制GSCs裸鼠移植瘤的生长，延长生存期的最佳方式。本项目的研究结果建立了以BACH1与FUS互作为起点的DLX6-AS1/miR-103a-3p为核心的调控GSCs生物学行为的新型分子网络，不仅为深入解析胶质瘤的发病机制提供了新的理论基础，也为恶性胶质瘤的综合治疗提供了新靶标。