细胞核体Cajal bodies和小分子非编码RNA在肿瘤干细胞中协同调节基因簇的形成

Nuclear bodies(NBs) are distinct subnuclear domains present in mammalian systems. Although numerous NBs, such as Cajal bodies(CBs), PML(promyelocyctic leukemia) NBs and IGC (interchromatin granule cluster) have been extensively studied, and changes in morphology and constitution of these nuclear bodies are associated with human diseases, the precise function and regulation of NBs are still poorly understood. Recently we have developed a six-color microscopic detection system and identified multiple small RNA genes to be clustered around CBs in cancer cells. Importantly, associations among these genes are not detectable in human primary diploid cells lacking CBs and they are impaired in cancer cells when CBs are specifically disassembled by RNAi. These data strongly suggest that CBs act as genome organizers which are able to recruit specific gene arrays and form gene clusters with the help of non-coding small RNAs, and thus likely regulate gene expression of specific regions in chromatin and influence gene function in cancer cells. To further analyze the role of CBs in the genome organization in cancer, we will develop a cancer stem cell model and perform genome-wide mapping of gene clusters associated with Cajal bodies and specific non-coding small RNAs by RNA-seq, small RNA-seq, chip-seq and ChIA-PET. In addition, we will also use siRNA and TALENs approaches to study the molecular mechanism of gene clusters associated with Cajal bodies and a possibly role in cancerogenesis.

细胞核体是一类独特的亚细胞区域。大量核体已被广泛研究，发现其形态和构成的改变与人类疾病密切相关，但其功能和调节目前仍不清楚。我们利用6D-FISH的检测系统，观察到细胞核体Cajal bodies（CBs）同时与多个小分子非编码RNA基因位点相互作用，形成基因簇，这种基因簇只存在于肿瘤细胞，原代细胞不存在。用siRNA破坏CBs的形成，基因簇也被破坏。结果提示， CBs很可能具有基因组组织者功能，在小分子非编码RNA协同下，募集特定基因，形成基因簇，调节染色体特定区域的基因表达，影响肿瘤细胞的功能。本项目拟建立肿瘤干细胞模型，利用RNA-seq, 小分子RNA-seq, Chip-seq, 研究长距离相互作用的新技术ChIA-PET在全基因组范围定位CBs和小分子非编码RNA相关的基因簇，并通过小分子RNA干扰, TALENs基因编辑技术研究基因簇形成的分子机制以及在肿瘤发生中的可能作用

细胞核体是一类独特的亚细胞区域。大量核体已被广泛研究，发现其形态和构成的改变与人类疾病密切相关，但其功能和调节目前仍不清楚。我们前期研究中利用6D-FISH的检测系统，观察到细胞核体Cajal bodies（CBs）同时与多个小分子非编码RNA基因位点相互作用，形成基因簇，这种基因簇只存在于肿瘤细胞，原代细胞不存在。用siRNA破坏CBs的形成，基因簇也被破坏。我们用染色质空间构象捕获4C实验证明， CBs很可能具有基因组组织者功能，在小分子非编码RNA协同下，募集特定基因，形成基因簇，调节染色体特定区域的基因表达，影响肿瘤细胞的功能。本项目通过建立肿瘤干细胞模型，利用CRISPR/Cas9基因编辑技术，RNA-seq, 小分子RNA-seq, 3'RACE， 染色质空间构象捕获4C 和Hi-C等 ，进一步阐明Cajal bodies通过小RNA介导形成基因簇，尤其是6号染色体的组蛋白基因簇，调节肿瘤细胞基因表达；并在全基因组范围定位CBs调节的基因簇及分子机制，以阐明CBs在肿瘤发生发展中的生物学功能及可能作用，为研究细胞核体提供新的理论依据。