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传递通过细胞外囊泡詹姆斯·帕顿（James G. Patton） - 调查员概括越来越多的证据支持了细胞外囊泡（EV）的假设通过蛋白质，RNA和脂质货物的转移的细胞 - 细胞通信形式。项目2 将专注于选择性EV miRNA转运和受体细胞吸收。我们以前表明来自KRAS突变细胞的电动汽车富含miR-100和miR-125b，我们有现在以3D培养在MiR-100和miR-125b中的EVS中显示受体细胞中的抗性。但是，尚不清楚mir-100和miR-125b（以及由于其他miRNA）被选择性地靶向分泌，而其他miRNA保留在细胞中。在这里，我们将专注于确定mir-100和miR-125b的转移是否可以改变肿瘤微环境使用斑马鱼中的新型体内异种移植模型。我们还将使用 CRISPR-Display的适应以检验特定RNA序列和/或碱基的假设修改调节选择性miRNA导出。相同的细胞培养分析将用于识别识别序列基序或修改的碱驱动特定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，我们目前的假设是转移 miR-100和miR-125b导致Wnt信号的激活，但mRNA的全范围这些miRNA的目标仍然未知。因此，我们将使用Rip-use组合使用差异表达分析的AGO2相关miRNA的免疫沉淀使用无偏序富集（RIP-使用），以识别miR-100和miR-125b的所有靶标。结肠隐窝中的正常细胞 - 细胞相互作用和干细胞生态位似乎是由分泌WNT和EGFR信号的相反梯度的电动汽车的分泌，我们的分析将识别潜在的治疗靶基因，其表达在适当的细胞细胞时会改变结直肠癌期间的沟通发生了改变。