Functional analysis of DDX1 in testicular germ cell tumors

细胞遗传学分析已经确定12p增益是人类睾丸生殖细胞肿瘤（TGCTs）中最常见的异常。有研究表明，在人类染色体12p区域，包括细胞周期蛋白d2在内的干细胞相关基因的扩增和过表达参与了生殖细胞肿瘤的发生。通过减法cDNA分析，我们发现DEAD box蛋白家族的成员Ddx1主要表达于小鼠胚胎的原始生殖细胞。用短干扰RNA敲低小鼠精原细胞GC-1spg中的Ddx1可抑制cyclin-D2、CD9和GDF3基因的表达。在小鼠周期蛋白- d2基因中，根据报告基因和凝胶转移实验，基因组DNA -348和-329区域负责DDX1的转录激活。同样，在人TGCT细胞系NEC8中，DDX1的敲低抑制了12p13.3染色体上的干细胞相关基因的表达，包括cyclin-D2、CD9和NANOG。ddx1敲除的TGCT细胞不能在裸鼠体内形成实体瘤。此外，原位杂交显示DDX1 mRNA在精原细胞瘤和非精原细胞瘤类型的人TGCT样本中均产生。我们得出结论，DDX1是睾丸肿瘤发生的关键因素。

Cytogenetic analysis has identified 12p gain as the most frequent abnormality in human testicular germ cell tumors (TGCTs). It has been suggested that amplification and overexpression of stem cell-associated genes, including cyclin-D2, on the human chromosome 12p region are involved in germ cell tumorigenesis. By subtractive cDNA analysis, we identified Ddx1, a member of the DEAD box protein family, as a gene predominantly expressed in the primordial germ cells of mouse embryos. Knockdown of Ddx1 in a mouse spermatogonia-derived cell line, GC-1spg, by short interference RNA repressed the expression of cyclin-D2, CD9 and GDF3 genes. In the mouse cyclin-D2 gene, genomic DNA region between -348 and -329 was responsible for transcriptional activation by DDX1 based on reporter and gel shift assays. Similarly, DDX1 knockdown in the human TGCT cell line NEC8 repressed the expression of stem cell-associated genes localized on chromosome 12p13.3, including cyclin-D2, CD9 and NANOG. DDX1-knocked-down TGCT cells could not form solid tumors in nude mice. Furthermore, in situ hybridization revealed that DDX1 mRNA was produced in both seminoma and non-seminoma types of human TGCT samples. We conclude that DDX1 is a critical factor for testicular tumorigenesis.