环状RNA—circCCNY促进恶性胶质瘤发生的作用机制研究

Malignant glioma patients have a poor prognosis and no effective treatment. Circular RNA plays an important role in the regulation of cellular biological behavior. However, the expression and function of circular RNA in glioma remains unclear. Our preliminary data showed that circ_0000235 (circCCNY) was up-regulated in glioma tissues compared to benign brain tumor tissues, this prompted that circCCNY may participate in glioma tumorigenesis. According to bioinformatic analysis, EIF4A3, a splicing factor obtaining potential binding sites with circCCNY, may promote cyclization and intracellular transporting of circCCNY in glioma. It is important that circCCNY overexpression enhanced the tumorigenesis rate and significantly elevated the viability of glioma cells, promoted cell proliferation and inhibited apoptosis, suggesting that circCCNY may play an important role in malignant glioma tumorigenesis. Gene Chips results and Gene Ontology analysis show that circCCNY may regulate its theoriotical downstream miR-16 gene cluster (miR-16-5p/miR-195-5p) to effect cyclin D、cyclin dependent kinase 4/6. We will utilize CRISPR/Cas9 system, adeno-associated virus, nude mice xenograft assay and so on to investigate the enrichment mechanism of circCCNY and its role on glioma oncogenesis. The present project aims to provide novel targets as diagnosis and gene therapy in glioma.

恶性脑胶质瘤患者预后差，尚无有效治疗方案。环状RNA在细胞生物学行为调控中发挥重要作用，然而在胶质瘤中表达及功能尚不清楚。申请者前期研究发现，恶性胶质瘤组织中环状RNA circCCNY表达水平较良性脑肿瘤显著升高，生物信息学分析表明剪接因子EIF4A3可能调控circCCNY在胶质瘤细胞中成环及转运。重要的是，过表达circCCNY增加胶质瘤成瘤率，增强胶质瘤细胞活力并抑制凋亡，提示circCCNY可能在恶性胶质瘤的成瘤过程中发挥重要作用。进一步miRNA芯片分析提示circCCNY可能通过调控miR-16基因簇进而调控周期蛋白cyclin D、CDK4/6表达。本研究拟通过CRISPR/Cas9基因沉默、腺相关病毒在体建模、裸鼠荷瘤等技术方法，探讨circCCNY在恶性胶质瘤中的重要功能及其作用机制，阐述环状RNA在恶性胶质瘤发生中的重要角色，为胶质瘤治疗提供新的策略。

环状RNA在细胞生物学行为调控中发挥重要作用，然而在胶质瘤中表达及功能尚不清楚。本研究采用RNA 免疫共沉淀、真核表达、荧光定量PCR、RNA干扰、荧光素酶报告基因、裸鼠移植瘤实验的方法，探讨环状RNA circCCNY在胶质瘤中参与肿瘤发生过程及机制。.本研究发现，胶质瘤组织中circCCNY较正常脑组织升高。过表达EIF4A3的U87及U251细胞中circCCNY的环状分子表达上升。提示EIF4A3能促进circCCNY成环。我们发现circCCNY过表达能引起胶质瘤细胞系增殖能力增强，miR-16-5p及miR-195-5p含量显著下降。荧光素酶实验证实circCCNY与miR-16-5p及miR-195-5p直接作用。miR-16-5p及miR-195-5p过表达能下调CDK4、CDK6及CCND含量。裸鼠移植瘤实验发现circCCNY沉默及miR-16-5p/miR-195-5p过表达均能抑制移植瘤生长，二者共同作用抑制肿瘤作用相协同。.我们的研究能得出如下结论：生理状态下正常脑细胞中circCCNY含量较低。在胶质瘤细胞中由于剪接因子EIF4A3表达升高，从而促使circCCNY成环，进而与抑癌基因miR-16-5p/miR-195-5p形成RNA沉默复合体，下调miR-16-5p/miR-195-5p表达含量。下调的miR-16-5p/miR-195-5p减轻了对于细胞周期相关因子CDK4、CDK6及CCND的抑制，加速了细胞增殖的进程。.通过本项目的系统研究，能够加深对胶质瘤发病机制的理解，为恶性胶质瘤预防和治疗的新靶点提供理论依据和实验基础。