miRNA-422a通过靶向调控PI3K-Akt信号通路抑制神经胶质瘤增殖、侵袭及转移

Studies reported that microRNA (miRNA) molecules take part in the process of Glioma cell proliferation, invasion and metastasis by regulate oncogenes, tumor suppressor genes and important cell signal transduction pathways. Our previous research showed the low expression of miR-422a molecules in glioma by using RT-PCR and Northern Blot technologies; overexpression of miR-422a suppressed the glioma cell proliferation, induce apoptosis and block cell cycle; meanwhile, the glioma cell invasion and metastasis capabilities in vitro was been depressed by miR-422a; we also has filtered 4 members of the PI3K-Akt signaling pathway as miR-422a downstream target gene by using cDNA chip and biological information methods. The propose of this study is to further confirm the direct target genes of miR-422a, explore the molecular mechanisms of miR-422a in glioma, and to verify the upstream regulatory mechanism in the process of miR-422a downregulation. This study will deeply explore the upstream and downstream regulation mechanism of miRNA-422a in glioma, and lay a theoretical foundation of deeply understanding of the role of miR-422a in the occurrence and development of glioma.

研究表明microRNA（miRNA）分子通过调控癌基因、抑癌基因及重要细胞信号通路参与神经胶质瘤的增殖、侵袭和转移。本项目前期研究发现miR-422a分子在神经胶质瘤中低表达；过表达miR-422a诱导神经胶质瘤细胞周期阻滞和凋亡，从而抑制增殖；同时过表达miR-422a抑制神经胶质瘤细胞的体外侵袭和转移能力；此外课题组基于cDNA芯片和生物信息学技术，明确了miR-422a下游靶基因包含有PI3K-Akt信号通路的四个关键分子。上述结果提示miR-422a与神经胶质瘤的增殖、侵袭和转移密切相关，并且提示miR-422a通过PI3K-Akt信号通路参与调控神经胶质瘤的发生发展。本课题组拟进一步确认其直接靶基因，进行功能分析，并研究miR-422a的上游调控机制。通过本研究，将有助于为以miRNA为靶点设计神经胶质瘤靶向药物提供理论线索和实验依据。

本项目在前期发现miR-422a在神经胶质瘤组织中低表达的基础上，旨在进一步探讨miR-422a对胶质瘤细胞恶性表型的影响，包括细胞活性、细胞增殖、细胞迁移及侵袭能力。并通过生物信息学分析筛选miR-422a的靶基因，而后利用EGFP融合报告载体技术结合实时定量PCR及Western blot方法进一步验证miR-422a对其靶基因的调控方式，并利用细胞表型实验确定靶基因对细胞恶性表型的影响，以阐明miR-422a作用于神经胶质瘤的分子机制。.通过研究我们证实与癌旁组织相比，miR-422a在胶质瘤组织中的表达水平明显降低，且在高分级瘤组织中的表达水平与低分级组织相比也明显降低。细胞表型实验结果显示，miR-422a在体外可抑制胶质瘤细胞U373及TJ905的细胞活性、增殖、迁移及侵袭能力。荷瘤鼠模型体内实验证实miR-422a在体内可抑制胶质瘤的增殖。通过生物信息学分析结合实时定量PCR及Western blot方法，最终确立PI3KCA为miR-422a的靶基因。接下来通过RNA干扰技术证实敲降PI3KCA的表达同样可抑制U373及TJ905的细胞活性、增殖、迁移及侵袭能力。挽救实验结果显示过表达PI3KCA可抵消miR-422a对胶质瘤细胞恶性表型的影响,进一步证实PI3KCA是miR-422a的直接作用靶基因。本研究确立了miR-422a对胶质瘤发生发展的影响及其分子机制，为理解神经胶质瘤的发生和开发胶质瘤早期诊断试剂及有效治疗药物提供实验线索和理论依据。