Effects of MYCN antisense oligonucleotide administration on tumorigenesis in a murine model of neuroblastoma
Effects of MYCN antisense oligonucleotide administration on tumorigenesis in a murine model of neuroblastoma
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DOI：
10.1093/jnci/djg045
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发表时间：
2003-09-17
期刊：
影响因子：
--
通讯作者：
Haber, M
中科院分区：
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作者：
Burkhart, CA;Cheng, AJ;Haber, M
背景人类MYCN（hMYCN）癌基因扩增是儿童神经母细胞瘤治疗失败的有力预测因子，hMYCN蛋白表达失调似乎与该疾病的发病机制密切相关。我们使用hMYCN反义（AS）寡核苷酸研究，在体外和体内，抑制hMYCN表达的治疗潜力。研究方法：我们用荧光标记的hMYCN AS或乱序（SCR）对照寡核苷酸瞬时转染具有扩增的hMYCN基因的人成神经细胞IMR-32细胞，并使用荧光激活细胞分选富集含有不同水平寡核苷酸的细胞群。我们使用荧光免疫细胞化学或逆转录聚合酶链反应来测定基因表达水平和台盼蓝拒染法来测定细胞群中的生长抑制。我们在hMYCN诱导的神经母细胞瘤的转基因小鼠模型（每组n = 20只小鼠）中检测了通过皮下植入的微渗透泵用AS或SCR寡核苷酸连续治疗6周对肿瘤生长的影响。所有统计检验均为双侧检验。结果如下：用AS寡核苷酸处理的IMR-32细胞的hMYCN蛋白和细胞增殖大约是SCR寡核苷酸转染或模拟转染对照的一半;差异具有统计学显著性。与SCR处理或未处理的对照小鼠相比，用AS寡核苷酸处理的转基因小鼠具有较低的肿瘤发病率和统计学显著较低的肿瘤质量。与对照组相比，AS寡核苷酸治疗在体外和体内与hMYCN和假定的hMYCN靶基因的表达降低，但与密切相关的基因的表达无关。几只AS利司那肽处理的小鼠在寡核苷酸给药部位的对侧发生肿瘤，而SCR利司那肽处理或未处理的小鼠显示双侧肿瘤生长。结论：使用AS寡核苷酸治疗可降低hMYCN蛋白的表达，即使在存在hMYCN癌基因扩增的情况下也是如此。体内靶向hMYCN癌基因的反义策略减少小鼠神经母细胞瘤的肿瘤发生研究其在儿童神经母细胞瘤中的临床效果是必要的。
Background. Human MYCN (hMYCN) oncogene amplification is a powerful predictor of treatment failure in childhood neuroblastoma, and dysregulation of hMYCN protein expression appears to be critically involved in the pathogenesis of this disease. We used hMYCN antisense (AS) oligonucleotides to investigate, both in vitro and in vivo, the therapeutic potential of inhibiting hMYCN expression. Methods: We transiently transfected human neuroblastorna IMR-32 cells, which have an amplified hMYCN gene, with fluorescently labeled hMYCN AS or scrambled (SCR) control oligonucleotides and used fluorescence-activated cell sorting to enrich for cell populations containing different levels of the oligonucleotides. We used fluorescence immunocytochemistry or reverse transcription polymerase chain reaction to assay gene expression levels and trypan blue exclusion to assay growth inhibition in the cell populations. We examined the effects of continuous treatment for 6 weeks with AS or SCR oligonucleotides via subcutaneously implanted microosmotic pumps on tumor growth in a transgenic mouse model of hMYCN-induced neuroblastoma (n = 20 mice per group). All statistical tests were two-sided. Results: IMR-32 cells treated with AS oligonucleotides had approximately half as much hMYCN protein and cell proliferation as either SCR oligonucleotide-transfected or mock-transfected controls; the differences were statistically significant. Transgenic mice treated with AS oligonucleotides had lower tumor incidence and statistically significantly lower tumor mass than SCR-treated or untreated control mice. Compared with control treatments, AS oligonucleotide treatment in vitro and in vivo was associated with decreased expression of hMYCN and putative hMYCN target genes but not with that of closely related genes. Several AS oligonucleotide-treated mice developed tumors contralateral to the site of oligonucleotide administration, whereas SCR oligonucleotide-treated or untreated mice displayed bilateral tumor growth. Conclusions: Decreased expression of hMYCN protein is achievable with the use of AS oligonucleotide treatment, even in the presence of hMYCN oncogene amplification. Antisense strategies targeting the hMYCN oncogene in vivo decrease mouse neuroblastoma tumorigenesis. Investigation of their clinical effect in children with neuroblastoma is warranted.