Gene profiling of cottontail rabbit papillomavirus-induced carcinomas identifies upregulated genes directly involved in stroma invasion as shown by small interfering RNA-mediated gene silencing
Gene profiling of cottontail rabbit papillomavirus-induced carcinomas identifies upregulated genes directly involved in stroma invasion as shown by small interfering RNA-mediated gene silencing
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DOI：
10.1128/jvi.78.14.7478-7489.2004
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发表时间：
2004-07-01
影响因子：
5.4
通讯作者：
Iftner, T
中科院分区：
文献类型：
--
作者：
Huber, E;Vlasny, D;Iftner, T
为了研究与恶性进展相关的细胞基因表达变化，我们在使用新西兰白兔的棉尾兔乳头瘤病毒（CRPV）鳞状癌模型中鉴定了差异表达基因。采用抑制减法cDNA杂交技术对crpv诱导的良性乳头瘤和癌mRNA分离物进行杂交，每对mRNA分离物均来自同一家兔。通过定量逆转录pcr （RT-PCR）和额外的活检进一步证实了23个缺失cdna的差异表达。8对乳头状瘤-癌对显示出持续上调的转录本的多功能性接头蛋白14-3-3 zeta和Y-box结合转录因子YB-1，而转录本的m型钙蛋白酶2和NB胸腺蛋白酶β，参与细胞运动和组织侵袭，以及酪蛋白激酶1 α，伴侣蛋白，和膜联蛋白1，被发现在大多数情况下上调。RNA-RNA原位杂交和激光捕获显微解剖结合定量RT-PCR分析证实了转录本在肿瘤细胞中的表达不受调控。相比之下，CRPV E7转录水平保持相当稳定，表明肿瘤诱导后不需要进一步上调E7的表达。在由新西兰大白兔角质形成细胞建立的crpv阳性细胞系中，小干扰rna介导的对编码YB-1、m型calpain 2或NB胸腺酶β基因表达的干扰导致基质室实验中细胞侵袭减少。
To investigate changes in cellular gene expression associated with malignant progression, we identified differentially expressed genes in a cottontail rabbit papillomavirus (CRPV) squamous carcinoma model employing New Zealand White rabbits. The technique of suppression subtractive cDNA hybridization was applied to pairs of mRNA isolates from CRPV-induced benign papillomas and carcinomas, with each pair derived from the same individual rabbit. The differential expression of 23 subtracted cDNAs was further confirmed by quantitative reverse transcription-PCR (RT-PCR) with additional biopsies. Eight papilloma-carcinoma pairs examined showed a constant upregulation of the transcripts for the multifunctional adaptor protein 14-3-3 zeta and the Y-box binding transcription factor YB-1, whereas transcripts for m-type calpain 2 and NB thymosin beta, which are involved in cell motility and tissue invasion, as well as casein kinase 1 alpha, chaperonin, and annexin 1, were found to be upregulated in the majority of the cases. RNA-RNA in situ hybridization and laser capture microdissection in combination with quantitative RT-PCR analysis verified the deregulated expression of the transcripts in the tumor cells. In contrast, CRPV E7 transcript levels remained rather constant indicating no requirement for a further upregulation of E7 expression following tumor induction. Small interfering RNA-mediated interference with expression of genes encoding YB-1, m-type calpain 2, or NB thymosin beta in a CRPV-positive cell line established from New Zealand White rabbit keratinocytes resulted in decreased cell invasion in matrigel chamber assays.