Functional delivery of viral miRNAs via exosomes
Functional delivery of viral miRNAs via exosomes
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DOI：
10.1073/pnas.0914843107
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发表时间：
2010-04-06
影响因子：
11.1
通讯作者：
Middeldorp, Jaap M.
中科院分区：
文献类型：
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作者：
Pegtel, D. Michiel;Cosmopoulos, Katherine;Middeldorp, Jaap M.
细胞和病毒来源的非编码调节性 microRNA (miRNA) 通过抑制 mRNA 翻译成蛋白质来控制基因表达。有趣的是，miRNA 通过称为“外泌体”的小囊泡主动分泌，这些小囊泡可以保护它们免受 RNase 的降解，这表明这些 miRNA 可能在产生它们的细胞外发挥作用。在这里，我们证明了 EBV 感染细胞分泌的 miRNA 被转移到未感染的受体细胞并在其中发挥作用。使用定量 RT-PCR 方法，我们证明成熟的 EBV 编码的 miRNA 是由 EBV 感染的 B 细胞通过外泌体分泌的。这些 EBV-miRNA 具有功能性，因为 MoDC 外泌体的内化会导致剂量依赖性、miRNA 介导的对已确认 EBV 靶基因的抑制，包括 CXCL11/ITAC（一种在原发性 EBV 相关淋巴瘤中下调的免疫调节基因）。我们证明，在 EBV 感染的 B 细胞的共培养过程中，EBV-miRNA 在未感染的邻近 MoDC 中积累，并表明这种积累是通过外泌体的转移介导的。因此，通过外泌体转移的外源EBV-miRNA被递送至受体细胞中基因抑制的亚细胞位点。最后，我们在 EBV 负载增加的患者的外周血单核细胞中发现，尽管 EBV DNA 仅限于循环 B 细胞群，但 EBV BART miRNA 存在于 B 细胞和非 B 细胞部分中，表明 miRNA 转移。总而言之，我们的研究结果与 miRNA 介导的基因沉默是一致的，它是免疫系统细胞之间细胞间通讯的潜在机制，可能被持久性人类 γ-疱疹病毒 EBV 利用。
Noncoding regulatorymicroRNAs (miRNAs) of cellular and viral origin control gene expression by repressing the translation of mRNAs into protein. Interestingly, miRNAs are secreted actively through small vesicles called "exosomes" that protect them from degradation by RNases, suggesting that these miRNAs may function outside the cell in which they were produced. Here we demonstrate that miRNAs secreted by EBV-infected cells are transferred to and act in uninfected recipient cells. Using a quantitative RT-PCR approach, we demonstrate thatmature EBV-encoded miRNAs are secreted by EBV-infected B cells through exosomes. These EBV-miRNAs are functional because internalization of exosomes by MoDCresults in a dose-dependent, miRNA-mediated repression of confirmed EBV target genes, including CXCL11/ITAC, an immunoregulatory gene down-regulated in primary EBVassociated lymphomas. We demonstrate that throughout coculture of EBV-infected B cells EBV-miRNAs accumulate in noninfected neighboring MoDC and show that this accumulation is mediated by transfer of exosomes. Thus, the exogenous EBV-miRNAs transferred through exosomes are delivered to subcellular sites of gene repression in recipient cells. Finally, we show in peripheral blood mononuclear cells from patients with increased EBV load that, although EBV DNA is restricted to the circulating B-cell population, EBV BART miRNAs are present in both B-cell and non-B-cell fractions, suggestive of miRNA transfer. Taken together our findings are consistent with miRNA-mediated gene silencing as a potential mechanism of intercellular communication between cells of the immune system that maybe exploited by the persistent human gamma-herpesvirus EBV.