Profiling of extracellular small RNAs highlights a strong bias towards non-vesicular secretion
Profiling of extracellular small RNAs highlights a strong bias towards non-vesicular secretion
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细胞外小 RNA 的分析凸显了对非囊泡分泌的强烈偏向
DOI：
10.1101/2020.12.01.406207
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Sork H
中科院分区：
文献类型：
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作者：
Sork H
细胞外环境由大量分子组成，包括可以与细胞外囊泡（EV）或可溶性蛋白复合物（非EV）结合分泌的细胞外miRNA。然而，对治疗性短RNA载体的兴趣主要在于EV，即传递绝大多数生物活性的载体。在这里，通过在亲本细胞中过表达miRNA和shRNA序列，并使用尺寸排阻液相色谱（SEC）将分泌组分离成EV和非EV部分，我们看到>98%的过表达的miRNA在非EV部分中分泌。此外，天然miRNA转录物的小RNA测序研究显示，尽管EV、非EV和亲本细胞中miRNA的丰度相关性良好（R2= 0.69-0.87），但在数量上，总miRNA的显著96.2-99.9%在非EV部分中分泌。然而，尽管EV仅含有一部分分泌的miRNA，但这些分子在37 °C下在含血清的环境中是稳定的，这表明如果实现足够的miRNA加载，EV可以在延长的时间段内保持递送能力。这项研究表明，被动内源性EV加载策略可能是一种相对浪费的将miRNA加载到EV的方法，并且未来需要主动miRNA加载方法来开发先进的EV miRNA疗法。
The extracellular environment consists of a plethora of molecules, including extracellular miRNA that can be secreted in association with extracellular vesicles (EVs) or soluble protein complexes (non-EVs). Yet, interest in therapeutic short RNA carriers lies mainly in EVs, the vehicles conveying the great majority of the biological activity. Here, by overexpressing miRNA and shRNA sequences in parent cells and using size exclusion liquid chromatography (SEC) to separate the secretome into EV and non-EV fractions, we saw that >98% of overexpressed miRNA was secreted within the non-EV fraction. Furthermore, small RNA sequencing studies of native miRNA transcripts revealed that although the abundance of miRNAs in EVs, non-EVs and parent cells correlated well (R2= 0.69–0.87), quantitatively an outstanding 96.2–99.9% of total miRNA was secreted in the non-EV fraction. Nevertheless, though EVs contained only a fraction of secreted miRNAs, these molecules were stable at 37 °C in a serum-containing environment, indicating that if sufficient miRNA loading is achieved, EVs can remain delivery-competent for a prolonged period of time. This study suggests that the passive endogenous EV loading strategy might be a relatively wasteful way of loading miRNA to EVs, and active miRNA loading approaches are needed for developing advanced EV miRNA therapies in the future.