长链非编码RNA介导miRNA的成熟对舌鳞癌细胞线粒体分裂和顺铂化疗敏感性的调控作用

Starting with the mitochondrial fission, the applicant has first revealed the regulatory role of mitochondrial fission in the cisplatin sensitivity of tongue squamous cell carcinoma, involving miRNAs as an important regulatory axis molecule, in recent years. The latest study demonstrating that long non-coding RNAs (lncRNAs) can mediate the mature process of miRNAs, raises the question that whether lncRNAs can affect the mitochondrial fission and cisplatin sensitivity in tongue squamous cell carcinoma via regulating miRNAs maturation. Given that our preliminary experiments have detected the regulatory function of lncRNA NR_029377 on the pre-miR-483 splicing to mature miR-483, we will screen the lncRNAs basing on regulation of the mitochondrial fission and cisplatin sensitivity in tongue squamous cell carcinoma by using lncRNA array in the present study. Then, to verify the biological functions of lncRNA-miRNA and miRNAs pathway, we will select key miRNAs by miRNA microarray following lncRNAs knockdown. And we will focus on the splicing process of pri(pre)-miRNAs mediated by lncRNAs in the nucleus and cytoplasm, thereby getting a comprehensive investigation of regulatory mechanism of lncRNAs on miRNAs maturation and providing a novel idea for the study on cisplatin sensitivity.

申请人近年以线粒体分裂作为切入点，首次发现线粒体分裂可调控舌鳞癌顺铂化疗敏感性，其中miRNA是重要的调控轴心分子。最新研究发现长链非编码RNA（lncRNA）可介导miRNA的成熟，那么lncRNA是否可通过调控miRNA的成熟进而影响舌鳞癌线粒体分裂和顺铂化疗敏感性呢？我们预实验初步发现lncRNA NR_029377可调控pre-miR-483剪切生成成熟的miR-483。因而本课题将基于lncRNA芯片筛选调控舌鳞癌线粒体分裂和顺铂化疗敏感性的功能lncRNA，对lncRNA干预后以miRNA芯片筛选其调控的下游关键miRNA，验明lncRNA-miRNA以及miRNA子通路对舌鳞癌细胞线粒体分裂和顺铂化疗敏感性的作用，机制研究聚焦于lncRNA介导miRNA前体结构在胞核和胞质中的剪切过程，从而全面剖析lncRNA对miRNA成熟的调控机制，为研究顺铂化疗敏感性提供新思路。

既往对逆转顺铂耐药尤其是获得性耐药的研究进展并不理想，因而探究顺铂化疗敏感性的上游机制可能更有现实价值。近年随着 RNA 组学研究的不断深入，发现非编码 RNA，尤其LncRNA，在肿瘤的多种生物学行为中发挥着重要的调控作用，也为研究顺铂化疗耐药机制提供了新的思路。本研究我们通过高通量的筛选，发现长链非编码RNA MPRL可调控线粒体分裂和顺铂的敏感性，且富集于细胞质并与pre-miR-483的环发生结合，并进而阻挡了TRBP对pre-miR-483的识别以及DICER对pre-miR-483的成熟剪切，进而影响了miR-483-5p的生成和下游FIS1介导的顺铂敏感性和线粒体分裂。同时我们还发现长链非编码RNA CISAL可通过特异的序列锚定于BRCA1的启动子上，并通过其5’端与BRCA1的转录因子GABPA特异结合，并将GABPA从BRCA1的启动子区域调离，抑制BRCA1的转录，进而介导下游miR-593-5p-MFF轴对线粒体分裂和顺铂敏感性的调控作用。我们采用高通量的方法，筛选出线粒体所富集的mitomiRs，并进一步发现其具有调控线粒体OXPHOS和糖酵解的功能。对其机制研究我们发现，mitomiR-2392可与线粒体基因组发生特异结合，并依赖于AGO2的作用，抑制线粒体基因的转录；mitomiR-5787可以靶向调控线粒体基因组MT-CO3促进其翻译；进一步的临床样本分析发现mitomiR-2392和mitomiR-5787以及调控的线粒体靶基因与铂类耐药性和预后相关。本课题从转录以及转录后调控水平阐明了长链非编码RNA可调控线粒体分裂的机制及其对顺铂化疗敏感性的作用，将为临床提高顺铂化疗敏感性提供新思路和更有效的干预靶点。