Identification of differentially expressed and developmentally regulated genes in medulloblastoma using suppression subtraction hybridization
Identification of differentially expressed and developmentally regulated genes in medulloblastoma using suppression subtraction hybridization
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DOI：
10.1038/sj.onc.1207475
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发表时间：
2004-04-22
期刊：
影响因子：
8
通讯作者：
Rutka, JT
中科院分区：
文献类型：
--
作者：
Yokota, N;Mainprize, TG;Rutka, JT
为了增加我们对髓母细胞瘤（MB）分子发病机制的理解，我们利用抑制减法杂交（SSH）技术来鉴定髓母细胞瘤中与小脑相比失调的基因。通过从相应的非肿瘤性小脑中减去共同的cDNA，从人和Ptch（+/-）杂合小鼠mb中获得了富含ssh的cDNA文库。人经典MB文库以人小脑总RNA作为对照组织；对于Ptch（+/-）杂合性MB，使用来自未受Ptch（+/-）影响的同窝的非肿瘤性小脑作为对照。通过对这些文库的差异筛选，我们分离了100多个上调的肿瘤cDNA片段，并利用NCBI BLAST程序对其进行了测序和鉴定。从中，我们选择了参与细胞增殖、抗凋亡和小脑分化的基因进行进一步分析。在人MB文库中发现的上调基因包括un33样蛋白（ULIP）、SOX4、Neuronatin （NNAT）、哺乳动物果蝇barh样1同源基因（BARHL1）、核基质蛋白NRP/B （ENC1）和同源盒OTX2基因。在小鼠MB文库中发现上调的基因包括cyclin D2 （Ccnd2）、thymopoietin （Tmpo）、Musashi-1 （Msh1）、蛋白磷酸酶2A抑制剂-2 （I-2pp2a）和Unc5h4(D)。利用半定量逆转录聚合酶链反应（RT-PCR），这些基因在人mb中的mRNA表达水平明显高于小脑。Western blot分析进一步证实了这些基因的一个子集在蛋白水平上的过表达。通过转染hes1 -荧光素酶，在表达高水平MSH1的TE671 MB细胞系中发现Notch通路过度活跃。这项研究揭示了一组可能参与MB发病机制的发育调节基因。
To increase our understanding of the molecular pathogenesis of medulloblastoma (MB), we utilized the technique of suppression subtractive hybridization (SSH) to identify genes that are dysregulated in MB when compared to cerebellum. SSH-enriched cDNA libraries from both human and Ptch(+/-) heterozygous murine MBs were generated by subtracting common cDNAs from corresponding non-neoplastic cerebellum. For the human classic MB library, total human cerebellar RNA was used as control tissue; for the Ptch(+/-) heterozygous MB, non-neoplastic cerebellum from an unaffected Ptch(+/-) littermate was used as the control. Through differential screening of these libraries, over 100 upregulated tumor cDNA fragments were isolated, sequenced and identified with the NCBI BLAST program. From these, we selected genes involved in cellular proliferation, antiapoptosis, and cerebellar differentiation for further analysis. Upregulated genes identified in the human MB library included Unc33-like protein (ULIP), SOX4, Neuronatin (NNAT), the mammalian homologue of Drosophila BarH-like 1(BARHL1), the nuclear matix protein NRP/B (ENC1), and the homeobox OTX2 gene. Genes found to be upregulated in the murine MB library included cyclin D2 (Ccnd2), thymopoietin (Tmpo), Musashi-1 (Msh1), protein phosphatase 2A inhibitor-2 (I-2pp2a), and Unc5h4(D). Using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), the mRNA expression levels for these genes were markedly higher in human MBs than in cerebellum. Western blot analysis was used to further confirm the overexpression of a subset of these genes at the protein level. Notch pathway overactivity was demonstrated in the TE671 MB cell line expressing high levels of MSH1 through HES1-Luciferase transfections. This study has revealed a panel of developmentally regulated genes that may be involved in the pathogenesis of MB.