High-throughput single-cell co-sequencing of small and large RNAs to identify molecular circuitry in cancer

对小 RNA 和大 RNA 进行高通量单细胞共测序，以识别癌症中的分子电路

基本信息

批准号：
10373050
负责人：
Rong Fan
金额：
$ 40.87万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-03-01 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10373050
关键词：
Acute Myelocytic Leukemia Address Adopted Advanced Development Bar Codes Basic Cancer Research Biochemical Biochemistry Cancer Biology Cancer Diagnostics Cells Classification Code Collaborations Coupled DNA Data Detection Development Devices Dimensions Discrimination Foundations Gene Expression Regulation Genetic Transcription Genomics Glass Heterogeneity Human Immobilization Informatics Letters Ligation Malignant Neoplasms Manuals Mediating Messenger RNA Methods MicroRNAs Microfluidics Modeling Molecular Motivation Mus Myeloid Leukemia Nature Oncogenic Pattern Performance Physicians Play Poly A Positioning Attribute Process Proteins RNA Reaction Reagent Reproducibility Research Scheme Scientist Slide Small Nucleolar RNA Small RNA Technology Therapeutic Time Transfer RNA Untranslated RNA Variant anticancer research base cancer cell cancer initiation cost empowered interest leukemia microdevice multiple omics neoplastic cell new technology new therapeutic target novel novel therapeutic intervention piRNA single cell technology technology development technology validation tool transcriptome tumor tumor heterogeneity tumor progression

项目摘要

PROJECT SUMMARY Small RNAs (smRNAs) are important non-coding regulators broadly implicated in human cancer initiation and progression. The functions and mechanism of smRNAs have been widely studied in cancer research. They are also being investigated as new therapeutic targets but the results so far are unsatisfactory, due in part to the complexity of miRNA-mRNA regulatory network coupled with cellular heterogeneity in human cancers. Thus, new tools that can co-profile smRNAs and large RNAs (lgRNAs) in large numbers of single cells can help address this long-standing challenge, add a new dimension to smRNA research, empower the discovery of new mechanisms of smRNAs to participate in cancer biology, and enable potential applications to cancer diagnostics and therapeutics. A close collaboration between PIs Fan and Lu has demonstrated, for the first time, co-sequencing of both smRNAs and lgRNAs from the same single cells (Wang et al., Nature Comm., 2019). It further showed that having paired smRNA and lgRNA profiles on the single-cell level can reveal miRNA-mediated gene regulation and new mechanisms for controlling miRNA expression and intercellular heterogeneity. However, this single-cell smRNA-lgRNA co-sequencing technology is a manual process with low throughput and high cost, limiting its potential for cancer research due to insufficient statistic power to interrogate highly heterogeneous tumor cells. In this application, we propose the advanced development of this technology to deliver a high-throughput single-cell smRNA/lgRNA co-sequencing (scSLRco-seq) technology. It employs a novel slip-transfer microdevice in combination with novel molecular barcoding scheme and downstream biochemistry for simultaneous analysis of smRNAs and lgRNAs in 1000’s of single cells. Specifically, we will (Aim 1) develop a microfluidic cross-flow patterning method to create 2,500 DNA barcode arrays for spatially-coupled capture and barcoding of small and large RNAs, (Aim2) develop a novel slip- transfer chip to integrate multi-step biochemistry workflow for high throughput scSLRCo-seq, and (Aim3) validate this technology using human and mouse myeloid leukemia models. This novel technology addresses the lack of capability for high-throughput single-cell smRNA/lgRNA co-profiling, filling a gap in single-cell omics field and enabling the study of new questions previously impossible to answer. It represents a major leap in the field and will find wide-spread use in cancer research and applications.

项目概要小RNA (smRNA) 是重要的非编码调节因子，广泛涉及人类癌症的发生和发展进展 smRNA 的功能和机制已在癌症研究中得到广泛研究。也作为新的治疗靶点进行研究，但迄今为止的结果并不令人满意，部分原因是 miRNA-mRNA 调控网络的复杂性与人类癌症中的细胞异质性相结合。可以在大量单细胞中共同分析 smRNA 和大 RNA (lgRNA) 的新工具可以提供帮助解决这一长期存在的挑战，为 smRNA 研究增添新的维度，促进发现 smRNA 参与癌症生物学的新机制，并使其在癌症中的潜在应用成为可能 Fan 和 Lu 之间的密切合作首次证明了诊断和治疗方面的优势。时间，对来自同一单细胞的 smRNA 和 lgRNA 进行共测序（Wang 等人，Nature Comm.， 2019）进一步表明，在单细胞水平上配对 smRNA 和 lgRNA 谱可以揭示 miRNA介导的基因调控以及控制miRNA表达和细胞间质的新机制然而，这种单细胞 smRNA-lgRNA 共测序技术是一个手动过程。通量低、成本高，由于统计能力不足，限制了其在癌症研究中的潜力在此应用中，我们提出了对高度异质性肿瘤细胞的高级开发。技术提供高通量单细胞 smRNA/lgRNA 共测序 (scSLRco-seq) 技术。采用新颖的滑移微器件与新颖的分子条形码方案相结合，下游生物化学，可同时分析 1000 个单细胞中的 smRNA 和 lgRNA。具体来说，我们将（目标 1）开发一种微流体错流图案化方法来创建 2,500 个 DNA 条形码用于小和大RNA的空间耦合捕获和条形码的阵列，（目标2）开发了一种新型滑动- 转移芯片集成多步骤生物化学工作流程以实现高通量 scSLRCo-seq，以及（Aim3）使用人类和小鼠骨髓性白血病模型验证该技术。缺乏高通量单细胞 smRNA/lgRNA 联合分析能力，填补了单细胞组学的空白领域并能够研究以前无法回答的新问题，这代表了该领域的重大飞跃。领域并将在癌症研究和应用中得到广泛应用。