Mechanisms and Functional Consequences of Selective miRNA transfer via extracellular vesicles

结直肠癌中的exRNA：生物发生和功能 项目2.通过以下途径选择性转移miRNA的机制和功能后果 胞外小泡 詹姆斯·G·巴顿--调查员 摘要 越来越多的证据支持这一假设，即细胞外小泡(EVS)构成一种新的 通过蛋白质、核糖核酸和脂类货物的转移实现细胞与细胞之间的通讯。项目2 将侧重于EV miRNA的选择性转运和受体细胞的摄取。我们之前 显示来自KRAS突变细胞的EV富含miR-100和miR-125b，我们已经 现在3D培养显示，富含miR-100和miR-125b的EV可以提供西妥昔单抗 受体细胞中的抗性。然而，目前尚不清楚miR-100和miR-125b(以及 与其他miRNAs一样)被选择性地作为分泌的靶标，而其他miRNAs保留在细胞中。 在这里，我们将重点确定miR-100和miR-125b的转移是否会改变 采用新型斑马鱼体内异种移植模型的肿瘤微环境。我们还将使用 CRISPR-Display的适应性，以检验特定RNA序列和/或碱基的假设 修饰调节选择性的miRNA输出。同样的细胞培养试验将被用于 识别识别序列基序的RNA结合蛋白(与项目1一致) 修饰碱基以促进特定miRNAs的分泌，然后将其扩展到体内 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信号的激活，但在整个mRNA范围内 这些miRNAs的靶标仍不清楚。因此，我们将使用RIP-Use组合 Ago2相关miRNAs的免疫沉淀及差异表达分析 无偏序列浓缩(RIP-USE)以识别miR-100和miR-125b的所有靶标。 结肠隐窝中正常的细胞-细胞相互作用和干细胞壁龛似乎是由 EV的分泌建立了Wnt和EGFR信号的相反梯度，我们的分析将 识别潜在的治疗靶基因，当适当的细胞-细胞时其表达发生改变 在结直肠癌期间，通讯会发生改变。

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.