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

• 批准号: 9898733
• 负责人: Rong Fan
• 金额: $ 41.71万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 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/9898733
• 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

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.

项目摘要 小型RNA（SMRNA）是在人类癌症开始和 进展。 SMRNA的功能和机制在癌症研究中已广泛研究。他们是 还被研究为新的治疗靶标，但到目前为止的结果不令人满意，部分原因是 miRNA-mRNA调节网络的复杂性以及人类癌中细胞异质性的结合。那， 可以在大量单细胞中串联SMRNA和大型RNA（LGRNA）的新工具可以帮助 应对这个长期存在的挑战，为SMRNA研究添加一个新的维度，增强发现的能力 SMRNA参与癌症生物学的新机制，并为癌症提供潜在的应用 诊断和治疗。 PIS Fan和Lu之间的密切合作证明了这一点 时间，同一单个单元的SMRNA和LGRNA的同时（Wang等人，自然通讯，， 2019）。进一步表明，在单细胞水平上具有配对的smRNA和lgRNA轮廓可以揭示 miRNA介导的基因调节和用于控制miRNA表达和细胞间的新机制 异质性。但是，这种单细胞smrna-lgrna共同测序技术是一个手动过程 低通量和高成本，由于统计能力不足而限制了其癌症研究的潜力 询问高度异质肿瘤细胞。在此应用程序中，我们提出了此类的高级发展 提供高通量单细胞SMRNA/LGRNA共同测序（SCSLRCO-SEQ）技术的技术。它 员工与新颖的分子条形码方案结合使用新颖的滑移微电位和 下游生物化学，用于对1000个单元中SMRNA和LGRNA的简单分析。 具体而言，我们将（AIM 1）开发一种微流体跨流图案方法来创建2,500个DNA条形码 用于空间耦合捕获和对大型RNA的条形码的阵列，（AIM2）开发了一种新型的滑动 - 转移芯片以整合高吞吐量SCSLRCO-SEQ的多步生物化学工作流程和（AIM3） 使用人和小鼠髓样白血病模型来验证这项技术。这项新技术讲话 缺乏高通量单细胞SMRNA/lgrna的能力，填充了单细胞OMICS的空白 现场并启用了以前无法回答的新问题的研究。它代表了 现场并将在癌症研究和应用中找到广泛的用途。