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草地贪夜蛾Sf9细胞外泌体microRNA调控AcMNPV增殖的研究

田伟彬, 张以农, 任菲菲, 严寄铭, 孙京臣

田伟彬, 张以农, 任菲菲, 等. 草地贪夜蛾Sf9细胞外泌体microRNA调控AcMNPV增殖的研究[J]. 华南农业大学学报, 2025, 46(2): 133-140. DOI: 10.7671/j.issn.1001-411X.202404027
引用本文: 田伟彬, 张以农, 任菲菲, 等. 草地贪夜蛾Sf9细胞外泌体microRNA调控AcMNPV增殖的研究[J]. 华南农业大学学报, 2025, 46(2): 133-140. DOI: 10.7671/j.issn.1001-411X.202404027
TIAN Weibin, ZHANG Yinong, REN Feifei, et al. Regulation of AcMNPV proliferation by exosomal microRNA in Spodoptera frugiperda Sf9 cells[J]. Journal of South China Agricultural University, 2025, 46(2): 133-140. DOI: 10.7671/j.issn.1001-411X.202404027
Citation: TIAN Weibin, ZHANG Yinong, REN Feifei, et al. Regulation of AcMNPV proliferation by exosomal microRNA in Spodoptera frugiperda Sf9 cells[J]. Journal of South China Agricultural University, 2025, 46(2): 133-140. DOI: 10.7671/j.issn.1001-411X.202404027

草地贪夜蛾Sf9细胞外泌体microRNA调控AcMNPV增殖的研究

基金项目: 中国博士后科学基金(2022M721207);国家自然科学基金(32202749);广东省自然科学基金(2023A1515011658)
详细信息
    作者简介:

    田伟彬,硕士研究生,主要从事昆虫细胞分子生物学研究,E-mail:tian46102256@163.com

    通讯作者:

    张以农: 孙京臣,教授,博士,主要从事昆虫细胞分子生物学及病毒结构学研究,E-mail:cyfz@scau.edu.cn

  • 中图分类号: Q966

Regulation of AcMNPV proliferation by exosomal microRNA in Spodoptera frugiperda Sf9 cells

  • 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。

    Abstract:
    Objective 

    To investigate the effect of Spodoptera frugiperda Sf9 cell exosomal microRNA (miRNA) on proliferation of Autographa californica multiple nucleopolyhedrovirus (AcMNPV).

    Method 

    Exosomes 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.

    Result 

    Exosomes 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.

    Conclusion 

    This 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.

  • 图  1   AcSw106-eGFP重组菌PCR鉴定

    M:DL2000 DNA marker;1~22:重组菌AcSw106-eGFP;23:阳性对照;24:阴性对照。

    Figure  1.   PCR identification of recombinant bacterium AcSw106-eGFP

    M: DL2000 DNA marker, 1−22: Recombinant bacterium AcSw106-eGFP, 23: Positive control; 24: Negative control.

    图  2   重组病毒AcMNPV-eGFP的鉴定

    Figure  2.   Identification of recombinant virus AcMNPV-eGFP

    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  4   Sf9细胞外泌体的粒径分析

    A:通过纯化产物的透射电镜图像进行细胞直径统计,B:对纯化产物进行纳米颗粒跟踪分析。

    Figure  4.   Particle size analysis of exosomes from Sf9 cells

    A: Cell diameter statistics through transmission electron microscopy images of purified product, B: Nanoparticle tracking analysis 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 sequence
    eGFP-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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出版历程
  • 收稿日期:  2024-04-15
  • 修回日期:  2024-05-06
  • 录用日期:  2024-05-16
  • 网络出版日期:  2025-01-19
  • 发布日期:  2025-01-20
  • 刊出日期:  2025-03-09

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