胶质瘤中一个功能未知的长链非编码RNA(HOXA11-AS)转录调控其邻居基因表达的新机制

Gliomas are the most common primary tumors in the brain. Patients diagnosed glioblastoma (GBM) have a median survival of approximately 1 year. We profiled the lncRNA expression signatures in 220 gliomas of Chinese patients, and identified that HOXA11-AS expression was closely associated with glioma grade, molecular subtype and KRAS/ERK pathway. Multivariate Cox regression analysis revealed that HOXA11-AS was an independent prognostic factor in GBM patients. However, the regulation mechanism of HOXA11-AS on its neighbor genes is currently unclear. Based on the high-throughput RNA sequencing (RNA-seq) of HOXA11-AS knock-in GBM cells and other glioma databases, we firstly plan to investigate the mechanism of HOXA11-AS may act as a competing endogenous RNA (ceRNA), effectively becoming a sink for let-7 family, thereby lead to the activation of KRAS/ERK signaling pathway. Secondly, we aim to demonstrate that HOXA11-AS and HOXA13 are controlled by an upstream promoter containing a conserved c-Myc and AP1 binding site via chromatin immunoprecipitation and luciferase reporter assay. The present study may provide insides for understanding the transcriptional regulation mechanism of KRAS/ERK pathway and HOXA11-AS may participate in a feedback loop that modulates neighbor protein coding genes, HOXA13 in glioma. Moreover, it will also provide rational theories and research methods for the mechanism of lncRNA.

课题组前期对 220 例胶质瘤患者临床资料和组织样本基因表达谱研究发现了一个尚未报道的长链非编码RNA(HOXA11-AS)。HOXA11-AS是国人胶质瘤独立预后因子，其表达与肿瘤级别和分子分型密切相关；同时还发现，HOXA11-AS与KRAS/ERK信号通路紧密相关。尽管已证实lncRNA对靶基因的调控机制，但其对邻居基因的转录调控机制至今尚未明确。因此本课题拟开展：1、应用RIP和荧光素酶技术解析 HOXA11-AS以内源性竞争RNA方式通过Let-7家族对KRAS/ERK信号通路的调控作用；2、应用ChIP和荧光素酶技术研究KRAS/ERK信号通路下游转录因子对HOXA11-AS及其邻居基因(HOXA13)的转录调控模式。本研究可揭示“胶质瘤中HOXA11-AS与KRAS/ERK通路存在自激活调控环路，并能转录调控其邻居基因”的新机制，为lncRNA机制研究提供理论基础和研究手段。

1.项目的背景.脑胶质瘤是最常见的原发性颅内肿瘤，世界卫生组织1998年公布按死亡率排序，恶性胶质瘤是34岁以下肿瘤患者的第2位死亡原因，是35-54岁患者的第3位死亡原因。国内外关于胶质瘤的治疗效果在近30年没有明显改进。基于此，应用不断创新的生命科学理论与新技术研究胶质瘤发生、进展及靶向治疗的机制，并进一步探索新的诊疗方法一直是当今神经外科领域的前沿课题。胶质瘤中信号通路和非编码RNA (non-coding RNA，ncRNA)的交互调控异常是发病的主要原因。.2.主要研究内容.① 研究胶质瘤中HOXA11-AS通过内源性竞争RNA的机制对抑癌基因Let-7家族进行负调控，进而激活KRAS/ERK信号通路；② 验证胶质瘤中KRAS/ERK信号通路下游转录因子(c-Myc)对HOXA11-AS的转录调控关系，建立“HOXA11-AS/KRAS/ERK/c-Myc/HOXA11-AS”自激活的调控环路；③ 验证胶质瘤中KRAS/ERK信号通路下游转录因子(AP1)对HOXA11-AS邻居基因(HOXA13)的转录调控关系，从而最终建立“HOXA11-AS/KRAS/ERK/c-Myc/HOXA11-AS自激活环路对HOXA13”的转录调控；④ 验证胶质瘤中HOXA11-AS通过激活MEK/ERK信号通路来影响胶质瘤的侵袭功能；⑤验证HOXA11-AS对胶质瘤干细胞活性的影响。.3. 重要结果.①“HOXA11-AS /KRAS/ERK/c-Myc/HOXA11-AS自激活环路对HOXA13”的转录调控；②阐述了HOXA11-AS对胶质瘤细胞细胞周期进展的调控机制；③阐述了HOXA11-AS对胶质瘤细胞侵袭能力的调控机制；④阐述了HOXA11-AS对胶质瘤干细胞活性的调控机制。.4. 关键数据及其科学意义.本项目揭示了胶质瘤中HOXA11-AS与KRAS/ERK通路存在自激活调控环路，并能转录调控其邻居基因(HOXA13)的机制，为探索胶质瘤的靶向治疗提供了参考。