LOC388955结合hnRNPH1调控Akt/mTOR通路促进胶质瘤增殖的机制研究

Glioma is of high morbidity and mortality. Recent studies showed that pseudogenes played vital roles in the tumorigenesis and tumor progression，However, the tumorigenesis of pseudogene LOC388955 in glioma remains elusive. Recently, we found that LOC388955 was up-regulated and correlated poor prognosis in glioma. LOC388955 promoted tumor proliferation in glioma. Further study showed that LOC388955 interacted with hnRNPH1, and knockdown of LOC388955 inactivated the Akt/mTOR signaling pathway. Based on the findings, we assume LOC388955 promotes glioma proliferation interacts with hnRNPH1 via Akt/mTOR signaling pathway. In this study, we aim to determine the expression of LOC388955 and hnRNPH1 and their clinical significance in glioma, to reveal the sites and mechanism of the binding between LOC388955 and hnRNPH1 and disclose the mechanism via which LOC388955-hnRNPH1-Akt/mTOR promotes cell proliferation in glioma, using a series of biological and immunological experiments in cell and animal models. Our study is likely to provide the reliable evidence of LOC388955 involved in the malignant process of glioma, and provide the scientific clue for the tumorigenesis and tumor progression in glioma.

胶质瘤恶性程度高，预后差。研究表明假基因在肿瘤的发生发展中发挥重要作用，但假基因LOC388955在胶质瘤中的作用及机制未明。我们前期研究发现：（1）LOC388955在胶质瘤组织中高表达且提示预后不良；（2）LOC388955促进胶质瘤细胞增殖；（3）LOC388955与hnRNPH1结合；（4）敲降LOC388955导致Akt/mTOR通路失活。因此，我们提出LOC388955通过结合hnRNPH1激活Akt/mTOR通路促进胶质瘤增殖的科学假说。本项目拟采用一系列免疫学及细胞生物学实验，在基因、蛋白表达及细胞功能上，明确LOC388955和hnRNPH1在胶质瘤中的表达及意义，揭示两者结合的机制，阐述LOC388955－hnRNPH1－Akt/mTOR通路促进胶质瘤增殖的作用及机制，从而获得LOC388955参与胶质瘤增殖的可靠证据，为诠释胶质瘤发生发展的分子机制提供科学依据。

研究显示假基因在胶质瘤中发挥重要的作用。该项目研究PREL1结构域包含蛋白1假基因6（PRELID1P6)在胶质瘤中的作用及机制。通过TCGA数据库筛选出异常表达的基因并通过功能筛选，发现PRELID1P6在胶质瘤中高表达，其高表达提示患者预后差。采用慢病毒转染并构建PRELID1P6稳定高、低表达的细胞株；采用小干扰RNA敲降PRELID1P6的表达；体内外实验发现PRELID1P6促进胶质瘤细胞增殖；采用RNA pulldown实验及质谱分析筛选与其相互作用的蛋白-hnRNPH1（heterogeneous nuclear ribonucleoprotein H1，核内不均一核糖核蛋白H1），并用RIP（RNA immunoprecipitation）及共定位实验验证两者的结合，同时探讨PRELID1P6作用于hnRNPH1的机制；挽救实验研究hnRNPH1在PRELID1P6促进胶质增殖中的作用；结果发现PRELID1P6与hnRNPH1结合并通过抑制泛素化而增强hnRNPH1的表达，挽救实验证明PRELID1P6促进胶质瘤细胞增殖的能力须依赖于hnRNPH1。转录组测序及基因集富集分析（Gene Set Enrichment, GSEA）方法预测PRELID1P6调控的下游通路，Western blot检测Akt/mTOR通路改变；采用挽救实验证实hnRNPH1在PRELID1P6调控Akt/mTOR通路中的作用；在过表达PRELID1P6的细胞中加入Akt/mTOR通路抑制剂MK2206，利用CCK8增殖实验发现PRELID1P6可激活Akt/mTOR信号通路，挽救实验证明PRELID1P6依赖于hnRNPH1激活Akt/mTOR信号通路，并通过激活Akt/mTOR信号通路促进胶质瘤细胞增殖。采用数据库预测与PRELID1P6结合的microRNA，筛选出调控PRELID1P6表达量的miR-1825，采用荧光素酶报告基因实验进行验证结果；miR-1825与PRELID1P6结合并负向调控PRELID1P6表达。本研究证明miR-1825-PRELID1P6-hnRNPH1-Akt/mTOR作用轴在胶质瘤的发生发展中发挥重要作用。