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

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

【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。

【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.