Mechanisms and Functional Consequences of Selective miRNA transfer via extracellular vesicles

通过细胞外囊泡选择性 miRNA 转移的机制和功能后果

基本信息

批准号：
10544797
负责人：
James G. Patton
金额：
$ 34.63万
依托单位：
VANDERBILT 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：
2020-01-22 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10544797
关键词：
Biogenesis Biological Assay CRISPR/Cas technology Cell Communication Cell Culture Techniques Cell secretion Cells Cetuximab Clustered Regularly Interspaced Short Palindromic Repeats Colon Carcinoma Colorectal Colorectal Cancer Communication Coupled Coupling Data Epidermal Growth Factor Receptor Epigenetic Process Genes Immunoprecipitation In Vitro KRAS2 gene Large Intestine Carcinoma Lipids Malignant Neoplasms Malignant neoplasm of pancreas Measures Mediating Messenger RNA Methods MicroRNAs Modification Mutation Neoplasm Metastasis Normal Cell Pattern Play Proliferating Proteins RNA RNA Sequences RNA-Binding Proteins Research Personnel Resistance Role Signal Transduction Techniques Testing Up-Regulation WNT Signaling Pathway Work Xenograft Model Zebrafish base cancer cell cell type colonic crypt differential expression exosome extracellular extracellular vesicles in vivo mutant novel pharmacologic rapid testing stem cell niche therapeutic target three dimensional cell culture transcriptome sequencing tumor growth tumor microenvironment uptake

项目摘要

exRNA in Colorectal Carcinoma: Biogenesis and Function Project 2. Mechanisms and Functional Consequences of Selective miRNA transfer via extracellular vesicles James G. Patton - Investigator Summary Increasing evidence supports the hypothesis that extracellular vesicles (EVs) constitute a novel form of cell-cell communication through the transfer of protein, RNA and lipid cargo. Project 2 will focus on selective EV miRNA transport and uptake by recipient cells. We previously showed that EVs from KRAS mutant cells are enriched in miR-100 and miR-125b and we have now shown in 3D culture that EVs enriched in miR-100 and miR-125b can confer cetuximab resistance in recipient cells. However, it remains unknown how miR-100 and miR-125b (as well as other miRNAs) are selectively targeted for secretion while other miRNAs are retained in cells. Here, we will focus on determining whether transfer of miR-100 and miR-125b can alter the tumor microenvironment using a novel in vivo xenograft model in zebrafish. We will also use an adaptation of CRISPR-Display to test the hypothesis that specific RNA sequences and/or base modifications regulate selective miRNA export. The same cell culture assay will be used to identify RNA binding proteins (in concert with Project 1) that recognize sequence motifs or modified bases to drive secretion of specific miRNAs which will then be extended to in vivo effects using the zebrafish xenograft model. Lastly, our current hypothesis is that transfer of miR-100 and miR-125b results in the activation of Wnt signaling but the full range of mRNA targets for these miRNAs remains unknown. Thus, we will use RIP-USE to combine immunoprecipitation of Ago2 associated miRNAs with differential expression analysis using Unbiased Sequence Enrichment (RIP-USE) to identify all targets of miR-100 and miR-125b. Normal cell-cell interactions and stem cell niches in the colonic crypt appear to result from the secretion of EVs that set up opposing gradients of Wnt and EGFR signaling, our analyses will identify potential therapeutic target genes whose expression is altered when proper cell-cell communication is altered during colorectal cancer.

结直肠癌中的 exRNA：生物发生和功能项目 2. 选择性 miRNA 转移的机制和功能后果细胞外囊泡詹姆斯·G·巴顿 - 调查员概括越来越多的证据支持这样的假设：细胞外囊泡（EV）构成了一种新的通过蛋白质、RNA 和脂质货物转移的细胞间通讯形式。项目2 将重点关注受体细胞的选择性 EV miRNA 转运和摄取。我们之前显示来自 KRAS 突变细胞的 EV 富含 miR-100 和 miR-125b，我们有现在 3D 培养显示富含 miR-100 和 miR-125b 的 EV 可以赋予西妥昔单抗受体细胞的抵抗力。然而，目前尚不清楚 miR-100 和 miR-125b（以及与其他 miRNA 一样）被选择性地靶向分泌，而其他 miRNA 则保留在细胞中。在这里，我们将重点确定 miR-100 和 miR-125b 的转移是否可以改变使用斑马鱼体内新型异种移植模型研究肿瘤微环境。我们还将使用一个改编 CRISPR-Display 来测试特定 RNA 序列和/或碱基的假设修饰调节选择性 miRNA 输出。相同的细胞培养测定将用于识别识别序列基序的 RNA 结合蛋白（与项目 1 一致）或修饰碱基以驱动特定 miRNA 的分泌，然后将其扩展到体内使用斑马鱼异种移植模型的效果。最后，我们目前的假设是 miR-100 和 miR-125b 导致 Wnt 信号传导激活，但全范围 mRNA 这些 miRNA 的靶标仍然未知。因此，我们将使用 RIP-USE 来结合使用免疫沉淀法对 Ago2 相关 miRNA 进行差异表达分析无偏序列富集 (RIP-USE) 用于鉴定 miR-100 和 miR-125b 的所有靶标。结肠隐窝中正常的细胞间相互作用和干细胞生态位似乎是由 EV 的分泌建立了 Wnt 和 EGFR 信号传导的相反梯度，我们的分析将识别潜在的治疗靶基因，当适当的细胞处理时，其表达会发生改变结直肠癌期间，沟通会发生改变。