烯壳铁氮磁珠介导Survivin ASO对肿瘤细胞的转染和抑制作用

doi:10.19586/j.2095-2341.2023.0058

生物技术进展 ›› 2023, Vol. 13 ›› Issue (5): 785-797.DOI: 10.19586/j.2095-2341.2023.0058

烯壳铁氮磁珠介导Survivin ASO对肿瘤细胞的转染和抑制作用

张晓旭¹(), 肖向茜¹^,², 潘逸群¹, 顾烨翔¹, 董礼¹, 党浩然¹, 康茜¹, 王明连¹^,²()

^1.北京工业大学环境与生命学部，北京 100124
^2.抗病毒药物北京市国际科技合作基地，北京 100124

收稿日期:2023-04-19 接受日期:2023-05-18 出版日期:2023-09-25 发布日期:2023-10-10
通讯作者: 王明连
作者简介:张晓旭 E-mail: 18332756281@163.com；
基金资助:
北京市自然科学基金面上项目(M22032)

Transfection and Inhibition of Tumor Cells by Survivin ASO Mediated by Graphene-shelled Ferro-nitride Magnetic Beads

Xiaoxu ZHANG¹(), Xiangqian XIAO¹^,², Yiqun PAN¹, Yexiang GU¹, Li DONG¹, Haoran DANG¹, Xi KANG¹, Minglian WANG¹^,²()

^1.Faculty of Environment and Life，Beijing University of Technology，Beijing 100124，China
^2.Beijing International Science and Technology Cooperation Base for Antiviral Drugs，Beijing 100124，China

Received:2023-04-19 Accepted:2023-05-18 Online:2023-09-25 Published:2023-10-10
Contact: Minglian WANG

摘要/Abstract

摘要：

基于石墨烯的生物相容性和对单链核酸的吸附性，研究烯壳铁氮磁珠(graphene-shelled ferro-nitride magnetic beads, GFeNMB)运载靶向Survivin的反义寡核苷酸(antisense oligonucleotide, ASO)及其对肿瘤细胞的抑制作用。首先用RNA Draw针对Survivin mRNA的二级结构设计ASO，合成FAM标记和未标记的ASO；基于石墨烯对单链核酸的吸附性和对荧光团的淬灭性，采用酶标仪检测荧光强度方法检测GFeNMB对ASO的吸附能力，并对GFeNMB和GFeNMB-ASO表征；磁极作用下将Survivin ASO磁转染至人非小细胞肺癌细胞A549中，荧光显微镜观察GFeNMB将ASO载入细胞的能力；ASO转染后，采用Western blot检测Survivinr的表达，活性氧（reactive oxygen species, ROS）试剂盒、流式细胞仪检测细胞凋亡，CCK-8和划痕实验检测细胞增殖和迁移能力。结果表明，GFeNMB对ASO具有良好的吸附性，GFeNMB与ASO混合20 min达最大吸附量(0.44 μg·mg^-1)；FAM-ASO经磁性载入细胞内呈绿色荧光且集中于细胞核，GFeNMB介导的核转染能力显著优于脂质体；ASO经磁转染至细胞后，Survivin蛋白的降低水平优于未处理组和脂质体转染组；磁转染Survivin ASO后，肿瘤细胞凋亡比例增大，细胞内ROS升高，细胞增殖和迁移能力受到抑制。综上，GFeNMB可将Survivin ASO转染至肿瘤细胞中，能够抑制细胞增殖，诱导细胞凋亡。GFeNMB-ASO磁转染细胞后下调靶基因表达的情况表明，GFeNMB有望成为单链寡核苷酸的转染介质。

关键词: 烯壳铁氮磁珠, Survivin, 反义寡核苷酸, 磁转染

Abstract:

Based on the biocompatibility and excel affinity for single-strand nucleic acids of graphene， we investigated the transfection ability of graphene-shelled ferro-nitride magnetic beads （GFeNMB） carrying the antisense oligonucleotides （ASO） to target Survivin， and the inhibitory effect on tumor cells. Firstly， the ASO sequences were designed based on the secondary structure of Survivin mRNA predicted by RNA Draw software， and FAM-labeled and unlabeled ASOs were synthesized. The affinity of GFeNMB for single-strand nucleic acids was detected based on fluorescence with FAM-ASO. Based on the adsorbability of graphene to single-stranded nucleic acid and the quenchability of fluorophore， the fluorescence intensity detection method was used to detect the adsorption capacity of GFeNMB to ASO， and GFeNMB and GFeNMB-ASO were characterized with transmission electron microscope and laser granulometer. Survivin ASO was magnetically transfected into human non-small cell lung cancer cell A549 with a magnetic pole. The changes of Survivin expression were tested by Western blot， apoptosis by reactive oxygen species （ROS） kit and flow cytometry， cell proliferation and migration ability by CCK-8 assay and scratch assay. The results confirmed the excel affinity of GFeNMB for ASO， and the maximum adsorption amount （0.44 μg·mg^-1） was reached after 20 min of mixing GFeNMB with ASO. FAM-ASO was magnetically transfected into cells， which was confirmed by intracellular fluorescence focused on the nucleus observed under confocal microscope. Moreover， GFeNMB mediated transfection into nucleus was significantly superior to the liposome transfection. The down-regulated level of Survivin protein expression after magnetic loading ASO was better than those of the untreated and liposome transfected groups， followed by an increase in the proportion of apoptotic cells， an increase of intracellular ROS level， and inhibition of cell proliferation and migration. The results suggested that GFeNMB could transfect Survivin ASO into tumor cells， thus inhibit tumor cell proliferation and induce apoptosis. The down-regulation of gene expression after GFeNMB-ASO magnetofection of cells also indicated that GFeNMB may be a useful transfection medium for single-strand oligonucleotides.

Key words: graphene-shelled ferro-nitride magnetic beads, survivin, antisense oligonucleotide, magnetofection

中图分类号:

Q291

张晓旭, 肖向茜, 潘逸群, 顾烨翔, 董礼, 党浩然, 康茜, 王明连. 烯壳铁氮磁珠介导Survivin ASO对肿瘤细胞的转染和抑制作用[J]. 生物技术进展, 2023, 13(5): 785-797.

Xiaoxu ZHANG, Xiangqian XIAO, Yiqun PAN, Yexiang GU, Li DONG, Haoran DANG, Xi KANG, Minglian WANG. Transfection and Inhibition of Tumor Cells by Survivin ASO Mediated by Graphene-shelled Ferro-nitride Magnetic Beads[J]. Current Biotechnology, 2023, 13(5): 785-797.

图/表 13

图1 37 ℃时Survivin mRNA的二级结构及ASO靶位

Fig. 1 Secondary structure of Survivin mRNA at 37 ℃ and the target sites of ASOs

图2 GFeNMB运载Survivin ASO磁转染肿瘤细胞过程

Fig. 2 Magnetofection of tumor cells with Survivin ASO carried by GFeNMB

图3 磁转染原理磁转染：基于GFeNMB外壳石墨烯的吸附性使ASO能与GFeNMB结合形成药物载体，此时FAM修饰的ASO荧光被猝灭。将药物载体加入培养的肿瘤细胞中，然后细胞培养板置于磁铁上，在培养箱中孵育40 min。磁场可以使药物载体快速沉积在转染细胞上，实现核酸的高效递送。转染数小时后，由于ASO与survivin mRNA交杂，荧光恢复。

Fig. 3 General mechanism of magnetofection

表1 反义寡核苷酸序列及其在Survivin mRNA上的靶位

Table 1 Sequences of antisense oligonucletides and their target sites on the Survivin mRNA

寡核苷酸	序列(5'→3')	Survivin mRNA上的靶位
ASO1^[32]	5'-CCCAGCCTTCCAGCTCCTTG-3'	232~251
ASO2	5'-CTCTATGGGGTCGTCATCTG-3'	255~274
ASO3	5'-TCTTGAATGTAGAGATGCGG-3'	100~119
ASO4	5'-CAAATCCATCATCTTACGCC-3'	1 491~1 510

图4 GFeNMB吸附Survivin ASO的质量和时间关系A：不同质量GFeNMB吸附Survivin ASO后剩余液体的荧光强度；B：不同时间GFeNMB吸附Survivin ASO后剩余液体的荧光强度

Fig. 4 Quality and time relationship of Survivin ASO adsorbed by GFeNMB

图5 GFeNMB与GFeNMB-Survivin ASO的TEM图A: GFeNMB；B: GFeNMB-Survivin ASO

Fig. 5 TEM images of GFeNMB and GFeNMB-Survivin ASO

图6 GFeNMB与GFeNMB-Survivin ASO的水相粒径分布

Fig. 6 Particle size distribution of GFeNMB and GFeNMB-Survivin ASO in water

图7 GFeNMB和脂质体对细胞的毒性作用

Fig. 7 Cytotoxicity of GFeNMB and liposome

图8 GFeNMB-Survivin ASO转染后细胞的共聚焦显微镜荧光结果A： GFeNMB-Survivin ASO转染A549细胞； B： Lip-Survivin ASO转染A549细胞； C： GFeNMB-Survivin ASO转染A549细胞后48 h。

Fig. 8 Fluorescence images of cells after GFeNMB-Survivin ASO transfection

图9 GFeNMB-Survivin ASO和Lip-Survivin ASO转染后Western blot检测蛋白表达A: Western blot检测各组细胞中Survivin的蛋白表达； B: Western blot灰度分析。ns表示与细胞组和脂质体组相比无显著性差异，****表示与细胞组和脂质体组相比差异在P<0.000 1水平有统计学意义。

Fig. 9 Protein expression of GFeNMB-Survivin ASO and Lip-Survivin ASO transfected by Western blot

图10 GFeNMB-Survivin ASO转染后细胞凋亡情况A：流式细胞仪检测细胞组和实验组的细胞凋亡；B：转染后24、48 h凋亡细胞比；C：GFeNMB-Survivin ASO转染后细胞内ROS水平。**、***分别表示与细胞对照组相比，差异在P<0.01、P<0.001水平有统计学意义。

Fig. 10 Apoptosis of cells after transfection with GFeNMB-Survivin ASO

图11 不同时间段不同序列GFeNMB-Survivin ASO转染后细胞活力

Fig. 11 Cell viability after transfection with GFeNMB-Survivin ASO with different sequences at different periods

图12 GFeNMB-Survivin ASO转染后划痕实验在0、24 h细胞迁移结果注：*、****分别表示与实验组之间和细胞组对照相比，差异在P<0.05、P<0.000 1水平有统计学意义。

Fig. 12 Cell migration results after transfected by GFeNMB-Survivin ASO at 0 and 24 h

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烯壳铁氮磁珠介导Survivin ASO对肿瘤细胞的转染和抑制作用

Transfection and Inhibition of Tumor Cells by Survivin ASO Mediated by Graphene-shelled Ferro-nitride Magnetic Beads

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