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

• 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
• 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

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.

exRNA在大肠癌中的生物学作用 项目2.选择性转移miRNA的机制和功能后果 细胞外囊泡 James G. Patton -研究员 总结 越来越多的证据支持细胞外囊泡（EV）构成一种新的 通过蛋白质、RNA和脂质货物的转移进行细胞间通讯的形式。计划2 将专注于受体细胞的选择性EV miRNA转运和摄取。我们之前 结果显示，来自KRAS突变细胞的EV富含miR-100和miR-125 b， 现在在3D培养中显示，富含miR-100和miR-125 b的EV可以赋予西妥昔单抗 受体细胞中的抗性。然而，仍然不清楚miR-100和miR-125 b（以及 如其它miRNA）被选择性地靶向分泌，而其它miRNA保留在细胞中。 在这里，我们将重点关注miR-100和miR-125 b的转移是否可以改变 肿瘤微环境，使用一种新的体内异种移植模型在斑马鱼。我们还将使用 修改CRISPR展示以测试特定RNA序列和/或碱基 修饰调节选择性miRNA输出。将使用相同的细胞培养试验， 识别识别序列基序的RNA结合蛋白（与项目1一致），或 修饰的碱基以驱动特异性miRNA的分泌，然后将其延伸到体内， 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-125 b导致Wnt信号转导的激活，但mRNA的全范围激活。 这些miRNAs的靶点仍然未知。因此，我们将使用RIP-USE来联合收割机 Ago 2相关miRNAs的免疫沉淀与差异表达分析 无偏序列富集（RIP-USE），以鉴定miR-100和miR-125 b的所有靶标。 正常的细胞间相互作用和结肠隐窝中的干细胞龛似乎是由 分泌的EV，建立Wnt和EGFR信号的相反梯度，我们的分析将 鉴定当适当的细胞-细胞间杂交时其表达改变的潜在治疗靶基因 在结直肠癌期间，沟通发生了变化。