Despite extensive research during the past decade elucidating the mechanism of RNA interference (RNAi) in insects, it is not clear how ingested or injected double-stranded RNA (dsRNA) triggers RNAi response in the whole body or even its progeny, which is referred to as systemic RNAi. In the present study, we aim to understand how the dsRNA delivered into cells causes systemic RNAi using Colorado potato beetle cells (Lepd-SL1). We first tested if dsRNA treatment induces systemic RNAi in Lepd-SL1 cells. Exposure of a new batch of Lepd-SL1 cells to the conditioned medium where Lepd-SL1 cells treated with dsRNA targeting inhibitor of apoptosis were grown for 6 h induced apoptosis in these new batch of cells. We hypothesized the exosomes in the conditioned medium are responsible for RNAi-inducing effect. To test this hypothesis, we isolated exosomes from the conditioned medium from Lepd-SL1 cells that had been treated with dsGFP (dsRNA targeting gene coding for green fluorescent protein) or dsLuc (dsRNA targeting gene coding for the luciferase) were grown. RNA present in the purified exosomes was analyzed to check if long dsRNA or siRNA is accumulated in them. The results from the electrophoretic mobility shift assay clearly showed that the long dsRNAs are present in the exosomes. By knockdown of candidate genes involved in endosome recycling and generation pathways, we found that Rab4 and Rab35 are involved in exosome production and transport.

尽管在过去的十年中进行了广泛的研究，阐明了昆虫中RNA干扰（RNAi）的机制，但尚不清楚摄入或注射的双链RNA（dsRNA）如何触发整个人体甚至其子代的RNAi反应。作为系统性RNAi。在本研究中，我们旨在了解使用科罗拉多马铃薯甲虫细胞（Lepd-SL1）传递到细胞中的dsRNA如何引起系统性RNAi。我们首先测试了dsRNA处理是否在Lepd-SL1细胞中诱导系统性RNAi。将新一批的Lepd-SL1细胞暴露于条件培养基，其中用靶向dsRNA的凋亡抑制剂处理的Lepd-SL1细胞生长6小时，在这些新批次的细胞中诱导凋亡。我们假设条件培养基中的外来体负责RNAi诱导作用。为了检验这个假设，我们从已经用dsGFP（编码绿色荧光蛋白的dsRNA靶向基因）或dsLuc（编码荧光素酶的dsRNA靶向基因）处理过的Lepd-SL1细胞的条件培养基中分离了外泌体。分析纯化的外泌体中存在的RNA，以检查其中是否积累了长dsRNA或siRNA。电泳迁移率变动分析的结果清楚地表明，长的dsRNA存在于外泌体中。通过敲除参与内体循环和生成途径的候选基因，我们发现Rab4和Rab35参与外泌体的产生和运输。分析纯化的外泌体中存在的RNA，以检查其中是否积累了长dsRNA或siRNA。电泳迁移率变动分析的结果清楚地表明，长的dsRNA存在于外泌体中。通过敲除参与内体循环和生成途径的候选基因，我们发现Rab4和Rab35参与外泌体的产生和运输。分析纯化的外泌体中存在的RNA，以检查其中是否积累了长dsRNA或siRNA。电泳迁移率变动分析的结果清楚地表明，长的dsRNA存在于外泌体中。通过敲除参与内体循环和生成途径的候选基因，我们发现Rab4和Rab35参与外泌体的产生和运输。