HULC-miR128-TNR轴调控PI3K/AKT信号通路促进胶质母细胞瘤恶性生长的分子机制

PI3K/AKT signaling pathway plays a pivotal role in Glioblastomas multiforme (GBM) via regulation of malignant growth and angiogenesis, exploration of the underlying mechanism presents as core issues in scientific research. LncRNAs and miRNAs are important regulator in post-transcription which attracted more and more attention recently. Our research suggested LncRNA HULC may competitively bind to miR128, and miR128 is a key molecular in GBM which regulates a vast spectrum of downstream genes including TNR, which serve as a precursor in PI3K/AKT signaling pathway. Our hypothesis is LncRNA HULC probably play as potential driven factor in GBM to promote malignant growth via activation of PI3K/AKT signaling pathway. Combining comprehensive scientific frontier and our previous study, we plan to further uncover this mechanism through several levels. (1) Molecular and cellular level: How LncRNA HULC and miR128 have properties and biology function. The network regulatory mechanism of PI3K/AKT signaling pathway was activated in post transcription and transcription level. (2) In vivo level: Evaluating intervention LncRNA HULC-miR128-TNR axis regulation function of PI3K/AKT signal pathway and the effect of treatment of GBM. (3) Human tissue level: Validation of LncRNA HULC-miR128-TNR as a PI3K/AKT status regulator and their correlation with pathology, clinical and prognosis in GBM. These findings will extend the understanding of GBM mechanism and provide new molecular markers and potential therapeutic targets of GBM.

PI3K/AKT是胶质母细胞瘤（GBM）恶性生长和血管形成的核心信号通路，研究其调控机制是重要的科学问题。LncRNAs和miRNAs是转录后调控的重要元件，前期研究发现LncRNA HULC竞争性结合miR128，miR128靶向调控PI3K/AKT信号通路的前驱分子TNR，故提出科学假设：HULC-miR128-TNR轴调控PI3K/AKT信号通路促GBM恶性生长。申请者拟深化研究：（1）分子细胞层面：确定LncRNA HULC和miR128共激活PI3K/AKT信号通路的调控机制；（2）整体动物层面：评估干预LncRNA HULC-miR128-TNR轴对PI3K/AKT信号通路调控功能和抗GBM细胞效应的影响；（3）人体组织层面：验证GBM组织中LncRNA HULC-miR128-TNR轴调控PI3K/AKT机制与病理、临床和预后的关联性。丰富GBM发展机制，挖掘潜在治疗新靶点。

胶质母细胞瘤（GBM）是成人中枢神经系统中一种极具侵袭性的恶性肿瘤，PI3K/AKT是胶质母细胞瘤（GBM）恶性生长和血管形成的核心信号通路，本研究通过细胞增殖、迁移、侵袭、细胞周期、凋亡实验、血管拟态实验等细胞功能实验，通过qRT-PCR、WB实验检测PI3K/AKT通路基因、EMT相关基因、凋亡相关基因的mRNA和蛋白表达水平，验证HULC和miR128 分别或协同通过PI3K/AKT通路促进GBM细胞增殖、迁移和侵袭作用，且它们也诱导肿瘤VM和EMT机制。通过蛋白质谱分析、生物信息学分析、双荧光素酶报告实验、CHIRP（chromatin isolation by RNA purification）等实验，验证了 HULC和miR128互作关系，并证实TNR为lncRNA HULC和miR-128的靶点，在分子层面验证了GBM组织中HULC和miR-128通过调控TNR激活PI3K/AKT信号通路的调控机制。通过BSP和WB检测PTEN启动子甲基化水平和乙酰化组蛋白H3 / H4表达水平，证实HULC降低PTEN启动子甲基化和组蛋白乙酰化水平，可促进胶质母细胞瘤增殖和侵袭的作用。通过构建并监测小鼠体内肿瘤异种移植模型、通过对263例GBM组织的NGS、原位杂交、免疫组化检测等技术，在动物层面和人体组织层面验证GBM组织中 HULC-miR128-TNR轴调控PI3K/AKT的机制，明确HULC与病理、临床和预后的关联性，丰富GBM进展的机制，认为HULC可作为GBM预后的分子靶标。