基于单细胞转录组测序的垂体腺瘤增殖异质性评价及与侵袭表型相关性研究

At present, little is known about the lineage differentiation and functional phenotype of the pituitary adenoma cells. Intratumoral heterogeneity that is genetically different subclones control disease progression. Our preliminary work revealed that proliferation-related gene expression promotes tumor progression, as well as the heterogeneity of the proliferative marker expression and ultrastructure of pituitary adenomas. The recurrent and primary pituitary adenomas with invasive subtypes were enrolled in the present research. We analyze gene expression of individual tumor cells at the transcriptome level using high-throughput single-cell RNA-seq. We aim to (1) explore the differentially expressed genes related to cell proliferation; (2) identify the cell subpopulation with common expression profiles by bioinformatics analysis and (3) explore the correlation between the expression of AURKA/NDC80 and the new subpopulation marker genes as biomarkers and the invasive phenotype of pituitary adenomas, and further to validate at the level of mRNA and protein. More important, the cell subpopulations with characteristics of stemness and their lineage differentiation could be identified by the transcriptional signatures analysis. The results of the study are expected to establish the subtype classification of pituitary adenoma based on single-cell transcriptome data, and to obtain the direct evidence that the proliferation heterogeneity of individual cell subpopulation causes tumor invasion and progression.

目前研究对垂体腺瘤细胞的谱系分化与功能表型所知甚少，肿瘤内异质性即基因学不同的亚克隆群调控疾病进程。我们前期工作发现增殖相关基因表达促进肿瘤进展，以及垂体腺瘤增殖标志物表达和超微结构的异质性。本课题以复发垂体腺瘤和原发侵袭性垂体腺瘤为研究对象，应用高通量单细胞RNA测序技术分析肿瘤个体细胞在转录组水平的基因型表达，着重于发现与细胞增殖相关的差异表达基因，应用生物信息学方法识别具有共同表达谱特征的细胞亚群，同时探索已知的增殖相关基因AURKA和NDC80与新型的细胞亚群标记基因作为生物学标志物与垂体腺瘤侵袭表型的相关性，并在核酸和蛋白水平验证；更为重要的是，通过转录组特征分析可能发现肿瘤内部具有干细胞特性的细胞亚群及其谱系分化特性。预期研究结果将建立基于单细胞转录组数据的垂体腺瘤亚型分型，获得肿瘤细胞亚群的增殖异质性与肿瘤侵袭和进展相关的直接证据。

背景：垂体腺瘤的生化特性和临床进程呈现复杂性与多样性，其它人体肿瘤常见的原癌基因突变、抑癌基因失活以及信号通路异常等在垂体腺瘤当中罕有表现，由于解剖位置难以到达无法进行垂体组织活检，也缺乏功能性人类垂体肿瘤细胞系以及可靠的动物模型，使垂体腺瘤的深入研究和临床治疗均面临挑战。.主要研究内容：1.垂体腺瘤单细胞cDNA文库构建。2.垂体腺瘤肿瘤间异质性。3.垂体腺瘤的瘤内异质性。4.关于肿瘤发生机制的探索.重要结果及关键数据：1.单细胞cDNA文库构建：1490个单细胞（来自16位垂体腺瘤患者）的mRNA信息，18个大块标本（来自18位垂体腺瘤患者）的mRNA信息 2.通过主成分分析（PCA）提出垂体瘤分类新标准:Pit-1阳性组/AR阳性组3.染色体拷贝数变异（CNV）分析发现垂体腺瘤常见染色体拷贝数变异位位点4.单细胞水平发现垂体腺瘤肿瘤内异质性的存在.科学意义：1.开创性的在单细胞水平进行垂体腺瘤的研究2.提出了垂体腺瘤分类新的标准3.单细胞水平验证垂体腺瘤染色体拷贝数变异情况4.单细胞水平验证了垂体瘤的瘤内异质性5.对垂体腺瘤的基础研究打开了新的思路。