胶质瘤线粒体特异性高表达的miR-92b-5p调控细胞衰老的作用机制研究

Cellular senescence is stable cell cycle arrest, which can inhibit tumor proliferation. In the process of cellular senescence, mitochondria is dysfunctioned which could paticipate in glioma initiation and development. Glioma is the most common nervous system tumor. The mean survival time is very short in high grades gliomas. Drug development targeting mitochondria will be a potential effect therapy for glioma. Mitochondrial DNA (mtDNA) is found to be mutated and dysregulated in glioma. Mitochondria-related miRNAs (mitomiRs) regulate the function of mitochondria by targeting mtDNA. MitomiRs express specially in cells and tissues, but their expression and function in glioma is unclear. In previous study, we have discovered that miR-92b-5p was the the most significant difference expressed mitomiR in glioma tissues and confirmed that suppression of miR-92b-5p could induce glioma cellular senescence. Therefore, we hypothesized that miR-92b-5p could inhibit glioma cellular senescence by regulating mitochondrial function. In this project, we planned to identify the mitomiR profile and screen out miR-92b-5p as a specifically overexpressed mitomiR in glioma, then to illustrate the effect and mechanism of miR-92b-5p on suppressing glioma cellular senescence by regulating mitochondrial function.

细胞衰老是稳定的细胞周期阻滞，可抑制肿瘤增殖。细胞衰老过程中伴随线粒体功能失调，参与胶质瘤发生发展。胶质瘤是最常见的神经系统肿瘤，高级别胶质瘤平均生存时间短，靶向线粒体的药物开发将成为有效治疗胶质瘤的方法。线粒体基因组DNA（mtDNA）在胶质瘤中突变、表达失调。线粒体中的miRNA（mitomiR）可通过靶向mtDNA调控线粒体功能，但mitomiR具有细胞及组织特异性，而胶质瘤中mitomiR的表达及功能暂未知。我们前期通过小RNA测序发现miR-92b-5p为胶质瘤中表达差异最显著的mitomiR，并证实抑制其表达可促进胶质瘤细胞衰老。因此，我们推测miR-92b-5p可能通过调控线粒体功能抑制胶质瘤细胞衰老。本项目拟通过鉴定胶质瘤mitomiR表达谱，筛选出特异性高表达的mitomiR——miR-92b-5p，阐明其通过调控线粒体功能抑制胶质瘤细胞衰老的作用及机制。

p53是胶质瘤中常见的突变基因，其突变与细胞代谢密切相关，代谢异常是胶质瘤发生发展的重要原因。miRNA参与胶质瘤中线粒体介导的细胞代谢，有研究证实线粒体中存在miRNA，但其在胶质瘤线粒体中的表达及功能不明。本项目通过小RNA测序筛选鉴定出胶质瘤p53野生型细胞U87和p53突变型细胞U251、U138和T98G线粒体中差异miRNA表达谱，发现miR-92b-5p在U251细胞线粒体中的表达显著高于U87细胞。采用构建线粒体特异性高表达miR-92b-5p载体、CRISPR-Cas9基因编辑、ChIP、LC-MS等方法研究发现，线粒体特异性高表达miR-92b-5p在U251细胞中促进增殖、转移和自噬，通过抑制线粒体DNA编码的ND5抑制NAD生成；转录组测序证实miR-92b-5p定位于胶质瘤细胞线粒体后可调控自噬、溶酶体功能和磷脂酰肌醇（GPI）生物合成信号通路相关基因LC3、p62、SLC11A2、PIGN及PIGN-227转录本的表达；突变型p53可增加胶质瘤细胞中miR-92b-5p的转录活性，不能促进其表达，却可促进其定位于线粒体中，其机制是p53突变后可影响mRNA生成、剪切相关蛋白与miR-92b-5p启动子结合；p53突变的U251细胞被编辑成野生型后，细胞中p62、SLC11A2和PIGN表达下降。于是，我们得出结论：胶质瘤细胞中p53突变影响mRNA生成、剪切相关蛋白与miR-92b-5p启动子结合，从而促进其定位于线粒体，进而通过抑制ND5的表达抑制NAD生成，通过介导p62、SLC11A2和PIGN的表达调控自噬、溶酶体功能和GPI合成。本项目的实施明确了p53突变影响胶质瘤中miR-92b-5p细胞内定位的机制，阐明了线粒体特异性高表达的miR-92b-5p在胶质瘤细胞中的作用及机制，为其成为潜在的治疗胶质瘤的靶点提供实验证据。