基于新方法开发的长非编码 RNA DANCR 在基因转录调控和骨肉瘤发病中的作用及分子机制研究

Osteosarcoma (OS) is the most common primary malignant bone tumor with high mortality in children and adolescents. However, there have been few studies on its molecular mechanism. Recent studies have shown that long non-coding RNA (lncRNA) is involved in tumor proliferation, metastasis and drug resistance, but its molecular mechanism is still unclear. LncRNA DANCR (Differentiation Antagonizing Non - Protein Coding RNA) participates in the regulation of cell differentiation, and its expression is significantly upregulated in a variety of cancers, including OS. Previous studies showed that DANCR was distributed in both cytoplasm and nucleus. Recently, it was found that in osteosarcoma, DANCR served as competing endogenous RNA (ceRNA) in the cytoplasm to play the post-transcriptional regulatory function, but the molecular mechanism by which it regulates gene expression in nucleus to affect the onset and development of osteosarcoma has yet to be elucidated.. This project aims to develop a novel method based on state-of-the-art CRISPR/Cas13d system to study the role of DANCR in regulation of target gene expression and the etiology of osteosarcoma. This project will conceptually advance our understanding of the OS pathogenesis and provide a basis for its diagnosis and treatment. The new method can also be widely applied to study the molecular function of lncRNA in various diseases or biological systems.

骨肉瘤（OS）是儿童和青少年死亡率相对较高的恶性骨肿瘤之一，但目前对其发病分子机制研究较少。近期研究表明，长链非编码RNA（lncRNA）参与肿瘤增殖、转移和耐药等过程，但其分子机制尚不明确。LncRNA DANCR参与细胞分化调控，其在包括OS在内的多种癌症中表达量显著上调。之前的研究表明DANCR在细胞质和细胞核内都有分布，最近有研究发现骨肉瘤中DANCR在细胞质中作为竞争性内源RNA（ceRNA）发挥转录后调控功能，但是其在细胞核内通过调控基因表达来影响骨肉瘤发生和发展的分子机制尚不清楚。.本项目旨在开发基于CRISPR/Cas13d系统的全新方法，来研究DANCR通过与基因组结合，重塑染色质环境来调控目的基因的表达及其在骨肉瘤发生发展中的分子机制。本研究将在分子水平加深对OS发病机制的认识，为其诊断和治疗提供基础，新方法也可以广泛用于研究lncRNA在各种疾病或生物系统中的功能。

本项目开发了一种基于CRISPR/Cas13d系统的新方法CARPID(CRISPR-assisted RNA-protein interaction detection method)，并使用该方法分别研究了XIST、MALAT1和DANCR等多个lnRNA的相互作用蛋白，为解析这些lncRNA的生物学功能及其在肿瘤尤其是骨肉瘤发生发展中的分子机制提供了新方法。本项目还使用HT-SELEX技术对丁香假单胞菌和铜绿假单胞菌的转录因子结合序列进行系统性研究，并建立了这两个物种基因表达调控网络。此外，本项目执行期间遭遇新冠大流行，我们使用HTR-SELEX技术对蝙蝠冠状病毒、MERS、SARS、新冠及其四个典型变种的核衣壳蛋白结合RNA序列的特异性进行了研究，发现N蛋白具有高度保守的RNA识别基序，这些结果为研究冠状病毒基因组组装提供新的见解，并且为新冠防治药物开发提供新思路，即可开发通过影响N蛋白识别病毒RNA从而影响病毒基因组组装的药物。