›› 2018, Vol. 61 ›› Issue (7): 795-800.doi: 10.16380/j.kcxb.2018.07.005

• 研究论文 • 上一篇    下一篇

草地贪夜蛾Sf9细胞中snoRNA Bm-15反义寡核苷酸的定位及其对Bm-15的干涉效率

李新梅, 邱妩洁, 崔斌, 杨宗霖, 申雅文, 路一平, 阚云超, 李丹丹*   

  1. ( 南阳师范学院, 河南省伏牛山昆虫生物学重点实验室, 河南南阳 473061)
  • 出版日期:2018-07-20 发布日期:2018-07-20

Cellular location of antisense oligonucleotide of snoRNA Bm-15 and its interference efficiency with Bm-15 in Spodoptera frugiperda Sf9 cells

LI Xin-Mei, QIU Wu-Jie, CUI Bin, YANG Zong-Lin, SHEN Ya-Wen, LU Yi-Ping, KAN Yun-Chao, LI Dan-Dan*   

  1.  (Henan Provincial Key Laboratory of Funiu Mountain Insect Biology, Nanyang Normal University, Nanyang, Henan 473061, China)
  • Online:2018-07-20 Published:2018-07-20

摘要: 【目的】利用反义寡核苷酸(antisense oligonucleotide, ASO)干涉昆虫核仁小RNA(small nucleolar RNA, snoRNA)Bm-15的表达,并探索ASO进入细胞后的代谢途径。【方法】利用脂质体携带Cy5标记的、2′-O核糖甲基化修饰和硫代磷酸骨架修饰的Bm-15 ASO转染草地贪夜蛾Spodoptera frugiperda Sf9细胞,然后通过Cy5标记与溶酶体及线粒体免疫荧光探针共定位研究Bm-15 ASO在细胞内的转运机制。利用实时荧光定量PCR检测转染Bm-15 ASO的Sf9细胞中Bm-15的表达量,分析ASO对snoRNA Bm-15的干涉效果。【结果】Bm-15 ASO转染草地贪夜蛾Sf9细胞48 h后,ASO荧光信号充斥整个细胞和位于细胞边缘的比例分别为34%和66%,说明ASO进入细胞后可能分布在不同的亚细胞器中;进一步对Bm-15 ASO和溶酶体及线粒体进行共定位发现,位于细胞边缘的ASO大多被运输至溶酶体而不会存在于线粒体等细胞器中。实时荧光定量PCR检测结果表明,转染Bm-15 ASO的Sf9细胞中Bm-15表达量下降了47%。【结论】即使经过多种化学修饰,ASO仍然逃避不了细胞内源降解机器的识别,这有效解释了某些情况下利用ASO干涉基因表达时靶标基因干涉效率较低的现象。

关键词: 昆虫, 草地贪夜蛾, 反义寡核苷酸, snoRNA, Bm-15, 溶酶体

Abstract: 【Aim】 To study the interference efficiency of antisense oligonucleotide (ASO) with insect small nucleolar RNA (snoRNA) Bm-15, and to investigate the intracellular delivery of ASO in cells. 【Methods】 Spodoptera frugiperda Sf9 cells were transfected with Cy5-labeled, 2′-O ribosomal methylation and phosphorothioate-modified ASO of Bm-15 through liposome. Co-localization of Bm-15 ASO (labeled with Cy5) and lysosome as well as mitochondria was screened by immunofluorescence. The interference efficiency of ASO with snoRNA Bm-15 was analyzed by detecting the expression level of Bm-15 in Sf9 cells transfected with Bm-15 ASO using real-time PCR. 【Results】 The proportions of ASO fluorescence signals inundating the whole cell and at the cell edge were 34% and 66%, respectively, in the transfected Sf9 cells after 48 h transfection of Bm-15 ASO, suggesting that ASO might be delivered into different subcellular organelles. Further co-localization analysis showed that Bm-15 ASO was transported into lysosomes but not mitochondria. qPCR result showed that the expression level of Bm-15 decreased by 47% in Sf9 cells transfected with Bm-15 ASO. 【Conclusion】 ASO can not escape from the cellular endogenous degradation machinery even after various chemical modifications, and this effectively explains the low interference efficiency of ASO with target gene in some circumstances.

Key words: Insect, Spodoptera frugiperda, antisense oligonucleotide, snoRNA, Bm-15, lysosome