Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in recipient cells. Argonaute 2 (Ago2) is a key miRNA-binding protein that has been identified in EVs and could influence RNA silencing. However, Ago2 is in a non-vesicular form in serum and can be an EV contaminant. In addition, RNA-binding proteins (RBPs), including Ago2, and RNAs are often minor EV components whose sorting into EVs may be regulated by cell signaling state. To determine the conditions that influence detection of RBPs and RNAs in EVs, we evaluated the effect of growth factors, oncogene signaling, serum, and cell density on the vesicular and nonvesicular content of Ago2, other RBPs, and RNA in small EV (SEV) preparations. Media components affected both the intravesicular and extravesicular levels of RBPs and miRNAs in EVs, with serum contributing strongly to extravesicular miRNA contamination. Furthermore, isolation of EVs from hollow fiber bioreactors revealed complex preparations, with multiple EV-containing peaks and a large amount of extravesicular Ago2/RBPs. Finally, KRAS mutation impacts the detection of intra- and extra-vesicular Ago2. These data indicate that multiple cell culture conditions and cell states impact the presence of RBPs in EV preparations, some of which can be attributed to serum contamination.

中文翻译：

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培养条件极大地影响细胞外囊泡制剂中囊泡和囊外 Ago2 和 RNA 的水平

细胞外囊泡 (EV) 携带的 miRNA 可以影响受体细胞中的基因表达和功能表型。Argonaute 2 (Ago2) 是一种关键的 miRNA 结合蛋白，已在 EV 中被发现，可以影响 RNA 沉默。然而，Ago2 在血清中呈非囊泡形式，可能是 EV 污染物。此外，包括 Ago2 在内的 RNA 结合蛋白 (RBP) 和 RNA 通常是次要的 EV 成分，其分类到 EV 中可能受到细胞信号传导状态的调节。为了确定影响 EV 中 RBP 和 RNA 检测的条件，我们评估了生长因子、癌基因信号传导、血清和细胞密度对小 EV (SEV) 中 Ago2、其他 RBP 和 RNA 的囊泡和非囊泡含量的影响准备工作。培养基成分影响 EV 中 RBP 和 miRNA 的囊内和囊外水平，其中血清对囊外 miRNA 污染有很大影响。此外，从中空纤维生物反应器中分离 EV 揭示了复杂的制剂，具有多个含有 EV 的峰和大量的泡外 Ago2/RBP。最后，KRAS 突变影响囊泡内和囊泡外 Ago2 的检测。这些数据表明多种细胞培养条件和细胞状态会影响 EV 制剂中 RBP 的存在，其中一些可归因于血清污染。