Regulation of AcMNPV proliferation by exosomal microRNA in Spodoptera frugiperda Sf9 cells
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摘要:目的
探究草地贪夜蛾Spodoptera frugiperda Sf9细胞外泌体microRNA(miRNA)对苜蓿银纹夜蛾核多角体病毒(Autographa californica multiple nucleopolyhedrovirus,AcMNPV)增殖的影响。
方法通过不连续蔗糖质量分数梯度超速离心纯化外泌体,随后利用透射电镜观察和纳米颗粒跟踪分析技术对纯化产物进行颗粒直径分析。运用sRNA高通量测序技术筛选AcMNPV感染Sf9细胞后外泌体差异表达的miRNA并预测其潜在靶基因对应的生物学通路。通过转染模拟物(mimic)和病毒感染等细胞试验以及qPCR验证外泌体miRNA的差异表达,并检测差异表达miRNA——sfr-miR-1a-3p对AcMNPV增殖的影响。
结果成功纯化Sf9细胞外泌体,且AcMNPV感染有效刺激Sf9细胞外泌体的分泌量增加。AcMNPV感染Sf9细胞72 h后,经与Rfam数据库比对,筛选出11个差异表达的外泌体miRNAs,其中,8个miRNAs的转录水平与测序结果趋势一致。通过预测miRNAs靶基因和KEGG富集分析,发现潜在的靶基因主要富集在cAMP信号通路、PI3K-Akt信号通路、癌症通路、人乳头瘤病毒信号通路等,这表明差异表达外泌体miRNA可能参与昆虫天然免疫反应。过表达sfr-miR-1a-3p能显著提高AcMNPV病毒vp39基因的表达。
结论本研究通过sRNA测序筛选了AcMNPV感染后Sf9细胞外泌体的差异表达miRNA,证明了AcMNPV通过促进外泌体分泌,协助被感染细胞传递miRNA−sfr-miR-1a-3p来影响周围未感染细胞,以促进自身增殖。以上结果为昆虫外泌体传递miRNA以调控病毒增殖的机制研究提供了重要的理论依据。
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关键词:
- 草地贪夜蛾 /
- Sf9细胞 /
- 苜蓿银纹夜蛾核多角体病毒 /
- 外泌体 /
- microRNA /
- sfr-miR-1a-3p
Abstract:ObjectiveTo investigate the effect of Spodoptera frugiperda Sf9 cell exosomal microRNA (miRNA) on proliferation of Autographa californica multiple nucleopolyhedrovirus (AcMNPV).
MethodExosomes were purified using discontinuous sucrose mass fraction gradient ultracentrifugation. Then the purified exosome samples were observed under transmission electron microscopy, and conducted particle diameter analysis using nanoparticle tracking analysis. By sRNA hight throughput sequencing analysis, exosomally differentially expressed miRNAs were identified and biological pathways corresponding to their potential target genes were predicted. Cellular experiments such as mimic transfection and virus infection, as well as qPCR were performed to verify the differential expression of exosomal miRNAs, and detect the effect of the differentially expressed miRNA of sfr-miR-1a-3p on the proliferation of AcMNPV.
ResultExosomes from Sf9 cells were successfully purified, and AcMNPV infection effectively stimulated exosome secretion from Sf9 cells. After 72 h of AcMNPV infection in Sf9 cells, a total of 11 differentially expressed miRNAs were identified in exosomal miRNAs comparing with the Rfam database, in which eight miRNAs’ transcription levels showed consistent trends with the sequencing results. Potential target genes were mainly enriched in the cAMP signaling pathway, PI3K-Akt signaling pathway, cancer pathway, and human papilloma virus signaling pathway, suggesting that differentially expressed exosomal miRNAs might participate in insect innate immune responses. Overexpressing sfr-miR-1a-3p significantly promoted the expression level of the AcMNPV gene vp39.
ConclusionThis study conducts sRNA sequencing to screen differentially expressed miRNAs in AcMNPV-infected Sf9 cell exosomes, demonstrating that AcMNPV promotes infection by enhancing exosome secretion and affecting surrounding uninfected cells via miRNA sfr-miR-1a-3p transfer. This research provides an important theoretical support for understanding the mechanism by which insect exosomal miRNAs regulate virus proliferation.
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图 3 Sf9细胞外泌体的纯化
A:Sf9细胞和被AcMNPV感染Sf9细胞荧光检测,B:通过蔗糖质量分数梯度离心纯化细胞外泌体,C:纯化产物的透射电镜观察。
Figure 3. Purification of exosomes from Sf9 cells
A: Fluorescence detection of Sf9 cells and Sf9 cells infected by AcMNPV, B: Purification of exosomes by sucrose mass fraction gradient centrifugation, C: Transmission electron microscopy observation of purified product.
图 5 AcMNPV-eGFP感染Sf9细胞后外泌体差异表达miRNA的验证
“**”“***”“****”分别表示在P<0.01、P<0.001和P<0.000 1水平差异显著(t检验)。
Figure 5. Validation of differentially expressed miRNA in exosomes of Sf9 cells infected by AcMNPV-eGFP
“**” “***” and “****” indicate significant differences at P<0.01, P<0.001 and P<0.000 1 respectively (t test).
图 6 sfr-miR-1a-3p对AcMNPV增殖的影响
A:sfr-miR-1a-3p 过表达分析,B:vp39在过表达sfr-miR-1a-3p的Sf9细胞中的表达量分析;“**”和“****”分别表示在P<0.01和P<0.000 1水平差异显著(t检验)。
Figure 6. Effect of sfr-miR-1a-3p on proliferation of AcMNPV
A: Overexpression analysis of sfr-miR-1a-3p, B: Analysis of vp39 expression in Sf9 cells overexpressing sfr-miR-1a-3p; “**” and “****” indicate significant differences at P<0.01 and P<0.000 1 respectively (t test).
表 1 本研究所用引物
Table 1 Primers used in this study
引物名称
Primer name引物序列(5′→3′)
Primer sequenceeGFP-Xma I-F AAACCCGGGATGGTGAGCAAGGGCGAGGA eGFP-Kpn I-R AAAGGTACCTTACTTGTACAGCTCGTCCATG novel-27614-qPCR-F GCGGACAATGGTGGCAA novel-30340-qPCR-F GCGTTAGGGAACCGAAGAAA novel-20036-qPCR-F CGAAAAGTCGGTGTGGCTGA novel-6941-qPCR-F CGCGAGCTAAGTCGAAATTTGTA sfr-miR-1a-3p-qPCR-F GCGCGTGGAATGTAAAGAAGT novel-3944-qPCR-F GCGCCATCCCTCACATGAT sfr-miR-10498-5p-qPCR-F CGCGTTGGTCAACGTTCAA novel-1523-qPCR-F CGCGGTCAGGTTGGCC novel-1841-qPCR-F CGCGGATGCGTCGAGTAG novel-37024-qPCR-F GCGCAGCCGAAACTGAAAT novel-4546-qPCR-F GCGCGCTCGTATATTAATTCTC vp39-qPCR-F TGATGCAAGCCGAACAGCTA vp39-qPCR-R GTGTTCGGGTTTGTGGTGTC Sf-GAPDH-qPCR-F TTGCTAACGTCTCGGTCGTC Sf-GAPDH-qPCR-R ATGACACGACCTGTTCCTCG -
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